Remedy or preventive for kidney disease and method of diagnosing kidney disease

ABSTRACT

A novel agent for therapy and/or prevention of kidney diseases as well as a diagnostic method (detection method) of kidney diseases is disclosed. The agent for therapy and/or prevention of kidney diseases comprises as an effective ingredient a substance which inhibits casein kinase 2. The diagnostic method of kidney diseases according to the present invention comprises measuring activity or content of casein kinase 2, or measuring expression amount of casein kinase 2 gene in a sample separated from body.

TECHNICAL FIELD

The present invention relates to an agent for therapy and/or preventionof kidney diseases, and to a method for diagnosis of kidney diseases.

TECHNICAL BACKGROUND

Kidney is a paired urinary organ located at backside in body cavity,which serve to keep homeostasis through 1) excretion of water, 2)excretion of metabolites, especially nitrogen components, 3) excretionof electrolytes, 4) excretion of foreign matters and 5) adjustment ofblood osmosis pressure, amount of body fluid and acid-base equilibrium,by generation of urine. Kidney is an organ having importantphysiological functions such as adjustment of blood pressure throughproduction and secretion of renin and prostaglandins, adjustment ofdifferentiation and maturation of erythrocytes in bone marrow throughproduction of erythropoietin, and activation of 25-hydroxyvitamin Dwhich is a precursor of vitamin D (Blood, 68 (Suppl), 170a, 1986, ProcNatl Acad Sci USA, 28, 1199, 1981). Kidney is an aggregate of about1,000,000 functional units called nephron, and nephron comprisesglomerulus, Bowman's capsule, proximal renal tubule, Henle's loop anddistal renal tubule. Nephrons join to a collecting duct, and thecollecting duct opens at renal pelvis. Glomerulus filters blood througha spherical agglomerate of blood capillaries to produce primitive urine.Primitive urine is subjected to reabsorption and secretion in renaltubules, thereby producing the final urine. Usually, glomeruli havecontrols such that necessary substances in the blood, especially serumproteins, do not leak during the filtration step. However, when theglomeruli are disordered, proliferation of mesangial cells, a kind ofthe constituting cells, and increase of the peripheral matrix occur, sothat the amount of proteins excreted to the urine is increased. Uponincrease of the amount of the excreted proteins in the urine, theproteins per se damage the renal tubules, which exacerbates the damageof the glomeruli. This vicious circle rapidly decreases the renalfunction.

Representative kidney diseases include glomerular and tubulointerstitialnephritis, and diabetic nephropathy. Acute nephritic syndrome, rapidlyprogressive glomerulonephritisglomerulonephritic syndrome, recurrent orsustained hematuria syndrome, chronic nephraticnephritic syndrome,diabetic nephropathy, lupus nephritis, IgA nephropathy, chronicpyelitis, nephrosclerosis, renal hypertension, gouty kidney, acute orchronic renal failure and so on are known. Although kidney diseasespresent complicated and a variety of pathoses, any of them may followsevere course reaching renal failure. Known therapeutic methods ofkidney diseases performed depending on the severity include therapiesfor retaining renal functions, such as rest cure, diet cure andpharmacotherapy; hemodialysis therapy and kidney transplantation. Uponreaching renal failure, dialysis therapy such as hemodialysis orperitoneal dialysis, or kidney transplantation is necessary. Althoughthe hemodialysis therapy is said to be the ultimate therapy of kidneydiseases, it merely eliminates wastes accumulated in the body due to therenal function disorder, and the function of severely damaged kidney isnot recovered, so that the patient has to continue the dialysis therapyaccompanying pain and troubles, which requires equipment and costs,throughout the life. Further, eventually, in most cases, heart failure,an infectious disease or the like is involved to death. Recently, thenumber of patients who start dialysis is increasing year by year, whichis problematic from the viewpoint of economy and society. Although theonly radical treatment of renal failure is kidney transplantation, sinceit has a number of problems including shortage of donors, difficultiesin tissue compatibility and avoidance of rejection reaction, the numberof cases is small. Thus, development of radical therapy or radicaltherapeutic agent is demanded. Although there are a number of diseasesand pathoses which cause renal failure, the most frequent causativediseases are nephritis and diabetic nephropathy. Therefore, to providetherapy and prevention of these kidney diseases is one of the mostimportant tasks in the field of kidney diseases. Nephritis includesglomerulonephritis starting from damage of glomeruli, and tubularnephritis starting from renal tubules. In the former case, deteriorationof renal tubules occurs following the damage of glomeruli, while in thelatter, the deterioration is limited to the renal tubules. Clinicallyimportant lupus nephritis, IgA nephritis and the like are included inglomerulonephritis, and its mechanism of onset is thought to includeimmunological mechanism and non-immunological mechanism. As an exampleof the former, deposition of an immune complex between an antigenoriginated from a bacterium such as staphylococcus or hemolyticstreptococcus, or a virus such as hepatitis B virus or measles, and anantibody thereto, is known. However, it is thought today that there area variety of causes, and there are many unsolved problems. By the retardof excretion of wastes in the body fluid due to sustained chronicnephritis, the burden to the kidney is increased, which furtherexacerbates the progress of nephritis, thereby progressing thedeterioration. The kidney in which the glomeruli and renal tubules arecompletely deteriorated to reach renal failure cannot recover by itself,and therapeutic method or therapeutic drug which may recover the kidneyhas not yet been completed. Although steroidal anti-inflammatory drugsare used against acute nephritis and light chronic nephritis, there areno radical therapeutic method against renal failure reached afteraggravation of chronic nephritis, and there is no way other than tocontinue dialysis therapy. However, the function of the kidney tosecrete hormones and the like is also very important. Especially, if theamount of erythropoietin produced at proximal renal tubules is decreaseddue to the deterioration of the proximal renal tubules, severe anemiaoccurs. Therefore, accurate diagnosis and therapy at an early stage aredemanded.

At present, for pharmacotherapy against nephritis, mainly steroidalanti-inflammatory drugs, as well as immunosuppressants, anti-plateletagents, antihypertensive agents, diuretic drugs and the like are useddepending on the cause or pathosis. For diabetic nephropathy, togetherwith strict glycemic control, antihypertensive agents such asangiotensin converting enzyme inhibitors, ATII receptor blockers andcalcium blockers are used. However, with these known therapeutic agentsagainst kidney diseases, it is difficult to sufficiently stop theprogress to renal failure, and the present state is far from the radicaltherapy (Hiroshi OKA and Osamu WADA responsible eds., “Medical ScienceUnabridged Dictionary”, Supplement 5, Latest Information of Therapies,p. 218, Kodansha, 1988). Thus, satisfactory therapeutic agent againstkidney diseases does not exist. Further, side effects accompanied bycontinuous long term use of the drugs are also problematic. Thus, atpresent, therapy and prevention of kidney diseases are basically carriedout by rest cure and/or diet cure. Pharmacotherapy against kidneydiseases is still in the process of trial and error, and development ofan effective drug is demanded.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide aneffective agent for therapy and/or prevention of kidney diseases, anddiagnostic method of kidney diseases.

For solving the above-described problems, the present inventorsintensively studied using the nephritis of rat models induced byantibody to glomerular basement membrane. That is, the present inventorssearched genes expressing in diseased kidney by comparing geneexpression in the diseased kidney and in the non-diseased kidney, todiscover that casein kinase 2 is prominently increased in the diseasedkidney. Further, the present inventors experimentally demonstrated thatkidney diseases may be diagnosed using the expression of casein kinase 2as an index, and that therapy and/or prevention of kidney diseases maybe attained by administration of a substance which inhibits caseinkinase 2, thereby completing the present invention.

That is, the present invention provides an agent for therapy and/orprevention of kidney diseases, comprising as an effective ingredient asubstance which inhibits casein kinase 2. The present invention alsoprovides a method for diagnosis of kidney diseases, comprising measuringactivity and/or content of casein kinase 2, and/or measuring expressionamount of casein kinase 2 gene in a sample separated from body. Thepresent invention also provides use of the substance which inhibitscasein kinase 2 for the production of an agent for therapy and/orprevention of kidney diseases. The present invention further provides amethod for therapy and/or prevention of kidney diseases, comprisingadministering an effective amount of the substance which inhibits caseinkinase 2 to a patient or an animal suffering from a kidney disease, orto human er an animal for which prevention of a kidney disease isdesired.

The agent for therapy and/or prevention of kidney diseases according tothe present invention is an excellent agent for therapy and/orprevention of kidney diseases for which sufficient effects are notexpected by the known drugs. Further, by the diagnostic method of kidneydiseases (detection method of kidney diseases) according to the presentinvention, pathoses of kidney diseases may be accurately and efficientlydiagnosed or detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows relationship between the expression amount of casein kinase2 gene in kidney tissue, i.e., in the cells constituting the kidney, andthe amount of excreted urinary protein, i.e., the state of kidneydisease.

FIG. 2 shows the effect of administration of an antisenseoligonucleotide against casein kinase 2α subunit on the amount ofexcreted urinary protein in nephritis rat models. *: p<0.05

FIG. 3 shows inhibitory action of apigenin against enzyme activity ofcasein kinase 2.

FIG. 4 shows the effect of administration of apigenin which is a caseinkinase 2 inhibitor on the amount of excreted urinary protein innephritis rat models. *: p<0.05

FIG. 5 shows the effect of administration of a casein kinase 2 inhibitoron the amount of excreted urinary protein in nephritis rat models. *:p<0.01

FIG. 6 shows the effect of administration of a casein kinase 2 inhibitoron the blood creatinine level in nephritis rat models. *: p<0.01

FIG. 7 shows the effect of administration of a casein kinase 2 inhibitoron the endogenous creatinine clearance in nephritis rat models. *:p<0.01

BEST MODE FOR CARRYING OUT THE INVENTION

The kidney diseases to be treated and/or prevented by the agent fortherapy and/or prevention of kidney diseases according to the presentinvention are not restricted, and examples thereof includeglomerulonephritis, interstitial nephritis, diabetic nephropathy, andchronic and acute renal failure. Further examples include acuteglomerulonephritis syndrome, acute progressive glomerulonephritissyndrome, recurrent or sustained hematuria syndrome, IgA nephropathy,chronic pyelitis, nephrotic syndrome, lupus nephritis, renalhypertension, gouty kidney, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, renal infarction, drug-inducednephropathy, dysfunction of transplanted kidney, renal function disorderaccompanied by renal calculus or ureteral obstruction. Among thesediseases, nondiabetic kidney diseases, inter alia, nephritis, arepreferred. Especially, the above-described diseases characterized byincrease of expression of casein kinase 2 and/or increase of enzymeactivity are preferred. The present invention may be applied not only tohuman, but also to mammalians such as simian, canine, feline, swine,bovine, ovine, goat, rabbit, rat and mouse. Thus, the agent according tothe present invention may be used not only as a human drug, but also asan animal drug. The agent for therapy and/or prevention of kidneydiseases is useful for therapy of kidney diseases, prevention for kidneydiseases, and for simultaneous therapy of kidney diseases and preventionof exacerbation of diseased state.

As mentioned above, the agent for therapy and/or prevention of kidneydiseases according to the present invention comprises as an effectiveingredient a substance which inhibits casein kinase 2. Casein kinase 2is a protein, and a kind of multifunctional protein kinase having a widerange of substrate specificity. It widely occurs in eukaryotic cells andexists in cytoplasms and nuclei. It consists of α subunit and α′ subunithaving a molecular weight of 41-44 kD, and β subunit having a molecularweight of 24-28 kD, and exists in the form of ααββ tetramer or αα′ββtetramer having a molecular weight of 130-140 kD (Biochemistry, 28,4072-4076(1989); Biochemistry, 29, 8436-8447(1990); Biochemistry, 28,9053-9058(1989)). The active center resides in the α subunit and α′subunit, and the enzyme is thought to phosphorylate serine and threonineresidues using ATP and GTP as the source of phosphate. However, up tonow, the significance of casein kinase 2 in the body and thesignificance in the diseased state have not been well understood.Further, it was not known that casein kinase 2 is expressed in diseasedkidney, and that kidney diseases may be cured and/or prevented byadministering a substance which inhibits casein kinase 2. The fact thatkidney diseases may be prevented and/or cured by inhibiting caseinkinase 2 is not at all expected from the prior art, and was firstdiscovered by the present inventors.

As will be concretely described in Examples below, the present inventorsdiscovered that the expression amount of casein kinase 2 gene in thekidney cells of diseased kidney was significantly larger than that innormal kidney cells, and that the diseased state of the kidney wassignificantly improved by administering a substance which inhibitscasein kinase 2. Therefore, the substance which inhibits casein kinase 2contained as an effective ingredient in the agent for therapy and/orprevention of kidney diseases according to the present invention may beany pharmaceutically acceptable substance which can inhibit caseinkinase 2. These substances are useful as agents for therapy and/orprevention of kidney diseases for which effective therapeutic method didnot exist. As will be described in Examples, in the cells constitutingthe kidney in diseased state, the expression amount of the casein kinase2 gene is significantly larger than in the cells constituting normalkidney. The larger the expression amount of the casein kinase 2 gene inthe cells constituting the kidney, the worse the state of the kidneydisease, and the closer the expression amount to the normal level, thecloser the state of the kidney disease to the normal state. This is notrestricted to the expression of the gene, but is also true for theexpression of the protein. As will be described in Examples, theexpression amount of the casein kinase 2 protein, that is, the contentof the protein, in the cells constituting diseased kidney issignificantly larger than that in the cells constituting normal kidney.The larger the expression amount, i.e., the content, of the caseinkinase 2 protein in the cells constituting the kidney, the worse thestate of the kidney disease, and the closer the expression amount, i.e.,the content, to the normal level, the closer the state of the kidneydisease to the normal state. Further, the expression amount, i.e., thecontent, of the casein kinase 2 protein is very closely related to theenzyme activity, i.e., the function, of casein kinase 2. That is, aswill be described in Examples, the larger the amount of the caseinkinase 2 protein, the higher the enzyme activity, i.e., the function, ofcasein kinase 2. These facts indicate that the larger the expressionamount of the casein kinase 2 gene, expression amount, i.e., thecontent, of the protein, and the enzyme activity, i.e., the function,the worse the state of the kidney disease, and the closer the amounts tothe normal levels by suppression of the expression, the closer the stateof the kidney disease to the normal state. Further, these facts give agenerally recognizable theoretical ground that any means for decreasingor inhibiting the expression of the casein kinase 2 gene, expressionamount, i.e., content of the protein, and/or enzyme activity, i.e., thefunction, as well as any substance which can decrease or inhibit thesehave actions or effects to make the state of the kidney disease close tothe normal state, so that they have actions or effects for curing andpreventing kidney diseases. For example, it is shown in Examples thatany of the various means for inhibiting casein kinase 2 (e.g., means forinhibiting expression of casein kinase 2 by using an antisenseoligonucleotide, and means for inhibiting enzyme activity of caseinkinase 2 by using a compound) has an action or effect to make the stateof the kidney disease close to the normal state, so that they haveactions or effects for curing and preventing kidney diseases. In otherwords, it is shown in Examples that any of the various substances havingfunctions to inhibit casein kinase 2 (e.g., a substance, antisenseoligonucleotide, inhibiting expression of casein kinase 2, and acompound inhibiting enzyme activity of casein kinase 2) has an action oreffect to make the state of the kidney disease close to the normalstate, so that they have actions or effects for curing and preventingkidney diseases. That is, it is proved that, in general, means andsubstances having actions to inhibit casein kinase 2 have actions tocure and prevent kidney diseases. Therefore, the substance whichinhibits casein kinase 2, which is contained as an effective ingredientin the agent for therapy and/or prevention of kidney diseases accordingto the present invention, may be any pharmaceutically acceptablesubstance which can inhibit casein kinase 2, and the substance is usefulas an agent for therapy and/or prevention of kidney diseases for whicheffective therapeutic method did not exist. Further, using theexpression of the casein kinase 2 gene, the expression amount, i.e., thecontent, and the enzyme activity, i.e., the function, as an index,kidney diseases may be diagnosed. Casein kinase 2 is known, and thoseskilled in the art can conceive the means and substances which inhibitcasein kinase 2, and these are also explained in detail in the presentinvention. Therefore, needless to say, any of these may be practiced bythose skilled in the art within the scope of usual techniques.

In the present invention, as the substance which inhibits casein kinase2 useful for the therapy and/or prevention of kidney diseases, anysubstance which results in the decrease of the effect of casein kinase 2may be employed. Preferred examples thereof include (1) substances whichinhibit expression of casein kinase 2 gene (substances which inhibittranscription, translation and/or modification, such as antisenseoligonucleotides, RNAis and ribozymes) and (2) substances which inhibitenzyme activity of casein kinase 2 (such as competitive andnon-competitive inhibitors against the enzyme activity, and anti-caseinkinase 2 antibody and antigen-binding fragments thereof). Substanceswhich decompose casein kinase 2 and substances which acceleratedecomposition of casein kinase 2 may also be used. Further, substanceswhich essentially inhibit or eliminate the signal transduction systemrelating to casein kinase 2 may also be employed. These inhibitorysubstances are those which inhibit or decrease the functional activityof casein kinase 2 determined by the method for measuring enzymeactivity concretely described in Examples below by at least about 30%,preferably by at least about 50%, more preferably by 80% or more. Themethod for measuring enzyme activity may be carried out withoutdifficulties according to the method commonly employed for usualmeasurement of enzyme activities. Concrete measuring method is describedbelow.

These casein kinase 2-inhibiting substances will now be described inmore detail.

Examples of the above-described (1) substances which inhibit expressionof casein kinase 2 gene include nucleic acids, compounds, peptides andthe like, which inhibit the expression (transcription, translation) ofthe gene or the protein of the subunit of casein kinase 2, or whichinhibit the post-translational modification thereof. Preferredsubstances are nucleic acids, and examples of the preferred nucleicacids include antisense oligonucleotides, RNAis and ribozymes againstthe casein kinase 2 gene. Further, vectors for gene introduction forgene therapy, containing an antisense gene against casein kinase 2 gene,cells in which the antisense gene against casein kinase 2 gene wasintroduced by the vector, and agents for gene therapy containing as aneffective ingredient the vectors for gene introduction for gene therapyor the cells in which the antisense gene against casein kinase 2 genewas introduced, are also included in these examples.

In normal cells, genetic information is transferred through the flow ofgenetic DNA-mRNA-protein. The nucleotide sequence of the mRNA coding forthe protein is called sense sequence, and the nucleotide sequencecomplementary to this sequence is called antisense sequence. Antisensesequences are explained as spontaneously generating biologicalsuppressors against gene expression, found in both prokaryotes (T.Mizuno, M-Y Chou and M. Inoue, Proc. Natl. Acad. Sci. USA, Vol. 81, pp.1966-1970, (1984)) and eukaryotes (S. M. Heywood, Nucleic Acids Res.Vol. 14, pp. 6771-6772 (1986)). These sequences are thought to functionto inhibit translation by being hybridized with the complementary mRNA(B. M. Paterson, B. E. Roberts and E. L. Kuff, Proc. Natl. Acad. Sci.USA, Vol. 74, pp. 4370-4374, (1977)). Oligonucleotides includingantisense oligonucleotides specifically hybridize with the complementarynucleotide sequence of any of precursor mRNA and mature mRNA. This termincludes naturally occurring oligomers consisting of bases, sugars andbonds between sugars (main chain), as well as oligomers havingnon-naturally occurring moieties, which function in the similar manner.In brief, antisense method is a method for nucleotidesequence-specifically inhibiting any of the expression of genetic DNA,function of mRNA or genetic DNA, translation to protein, transportationto cytoplasm, and optional activity necessary for the total biologicalfunctions by chemically synthesizing an antisense oligonucleotide havinga nucleotide sequence complementary to mRNA transcribed from a geneticDNA, administering the antisense oligonucleotide to cells or individualso as to make it specifically bind with the complementary single- ordouble stranded mRNA to form double-stranded structure. By this, sinceall or a part of the functions of the mRNA or the genetic DNA cannot becarried out, defect of a part of the genome regulating proper expressionof the protein is resulted, and the flow of the genetic information fromthe genetic DNA to the protein is blocked. As a result, the expressionof the gene is regulated in the negative direction and inhibited.Antisense method nucleotide sequence-specifically inhibits theexpression of the gene having the same sequence as the antisenseoligonucleotide. Many recent studies proved that the antisenseoligonucleotides are useful for excellent methods for analyzing genefunction (Rothenberg et al., J. Natl. Cancer Inst. 1989, 81, 1539-1544,Zon, G. Pharmaceutical Res. 1988, 5, 539-549).

Since an antisense oligonucleotide is a long chain composed of fourtypes of base units, the antisense oligonucleotide against any targetgene may easily be synthesized. By virtue of the recent progress ofoligonucleotide chemistry, and by virtue of the recent progress in thesyntheses of nuclease-resistant oligonucleotides, S-oligonucleotides andthe like, use of antisense oligonucleotides as a novel therapeuticmethod can be devised now. Cells contain various types of exo- andendonucleases which can decompose nucleic acids. A number ofmodifications of nucleotides and nucleosides have been shown to giveresistance to digestion by nucleases when compared with naturallyoccurring oligonucleotides. Such modified or substitutedoligonucleotides are often preferred to those of the natural form.Nuclease resistance is routinely measured by incubating anoligonucleotide with a cell extract or isolated nuclease solution, andmeasuring the remaining intact oligonucleotide with time usually by gelelectrophoresis. An oligonucleotide so modified as to promote thenuclease resistance remains intact for a longer time than the unmodifiedoligonucleotide. Various modifications of oligonucleotides for promotingor giving nuclease resistance are known. Examples of the intendedpreferred modes of modification of oligonucleotide include thoseemploying phosphorothioates as in S-oligonucleotides and those employingbonds between sugars through, phosphotriester, methyl phosphonate, shortchain alkyl, cycloalkyl, short chain hetero atoms, or heterocyclicstructure. S-oligonucleotide (nucleotide phosphorothioate) is anisoelectric analogue of an oligonucleotide (O-oligo) in which the oxygenatoms in the phosphate group, which oxygen atoms do not participate inthe polycondensation, are substituted by sulfur atoms. S-oligonucleotidemay be obtained by treating the corresponding O-oligo with3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur-transferringreagent. See R. P. Iyer et al., J. Org. Chem., Vol. 55, pp. 4693-4698,(1990); and R. P. Iyer et al., J. Am. Chem. Soc. Vol. 112, pp.1253-1254, (1990). The oligonucleotides having morpholino main chainstructure are also preferred (Summerton, J. E. and Weller, D. D., U.S.Pat. No. 5,034,506). In another preferred mode, the phosphodiester mainchain of the oligonucleotide may be substituted by polyamide main chainsuch as protein-nucleic acid (PNA) main chain (P. E. Nielsen, M. Egholm,R. H. Berf, O. Buchardt, Science 1991, 254, 1497). In this case, eachbase is directly or indirectly bound to the aza nitrogen atom in thepolyamide main chain. Oligonucleotide may have a sugar mimic such ascyclobutyl in place of pentofuranosyl group. Further, typically, theoligonucleotide has at least one modified nucleotide region which givesone or more advantageous properties (e.g., promotion of the nucleaseresistance, increase in the uptake into cells, and increase in bindingaffinity to RNA target) and a region serving as a substrate for thecleavage by RNaseH. In a preferred mode, the oligonucleotide has atleast one region modified for increasing the binding affinity to thetarget, and usually has a region serving as a substrate of RNaseH. Theaffinity to the target (in this case, the nucleic acid coding for caseinkinase 2) is routinely determined by measuring the melting temperature(Tm) of the oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and the target dissociate. The dissociation isdetected spectrophotometrically. The higher the melting temperature(Tm), the higher the affinity of the oligonucleotide to the target. In amore preferred mode, the region in the oligonucleotide, which ismodified for increasing the binding affinity to the mRNA of caseinkinase 2 comprises an at least one nucleotide of which 2′-position ofthe sugar is modified (2′-O-alkyl- and 2′-fluoro-modifiedoligonucleotides are most preferred). Such a modification is routinelyincorporated into the oligonucleotide, and the modified oligonucleotideexhibits higher melting temperature (Tm) (i.e., higher binding affinityto the target) than that of 2′-deoxyoligonucleotide. The resultingincreased affinity very much promotes the effect of the antisenseoligonucleotide against casein kinase 2. RNaseH is a cellularendonuclease which cleaves RNA chains in RNA:DNA duplexes. Therefore,activation of this enzyme results in the cleavage of the RNA target, andmay very much promote the efficiency of the antisense inhibition.Cleavage of the RNA target may be routinely shown by gelelectrophoresis.

Oligonucleotide or its analogue preferably contain about 12 to about 50,more preferably 12 to 25, still more preferably 16 to 22 bases. Further,such an oligonucleotide or its analogue specifically hybridizes with thetarget mRNA nucleotide sequence under physiological conditions (meltingtemperature (Tm) of substantially higher than 37° C., preferably atleast 50° C., typically at 60° C. to 80° C. or higher). Thehybridization preferably corresponds with the stringent hybridizationconditions, and the conditions are so selected as to attain atemperature which is lower than the melting temperature (Tm) with thetarget mRNA nucleotide sequence at the ion strength and at the pH byabout 10° C., preferably by about 5° C. Further, complementarity (thedegree of complementariness of a polypeptide to another polypeptide) maybe quantified by the ratio of bases expected to form hydrogen bonds eachother according to the generally accepted base-paring rule. The terms“specifically hybridizable” and “complementary” are the terms used fordescribing the degree of complementarity which is sufficient to attainstable and specific binding between the target DNA or RNA and theoligonucleotide. It should be understood that 100% complementarity isnot necessary to be able to specifically hybridize. However, 100%complementarity is most preferred for the specificity and goodtranslation inhibition. Even in cases where the complementarity is not100%, the complementarity is preferably not less than 90% (that is, thenumber of bases which mismatch with the bases of mRNA to be paired inthe entire antisense oligonucleotide to be hybridized with the mRNA isnot more than 10%). In cases where the binding of the oligonucleotide tothe target interferes the normal function of the target molecule so asto eliminate the utility of the target molecule, and in cases where theoligonucleotide has a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to the non-targeted sequencein the conditions under which the specific binding is desired, forexample, under the physiological conditions in case of therapeutictreatment, the oligonucleotide is specifically hybridizable. Such anoligonucleotide or its analogue may be prepared simply and routinely bythe well-known solid phase synthesis. As for the studies of chemicalsynthesis of oligonucleotides, see Goodchild, Bioconjugate Chemistry,Vol. 1, pp. 165-167, (1990). For example, oligonucleotides aresynthesized by an automated DNA synthesizer (Applied Biosystems Model380B) using the standard phosphoroamidite chemistry in which oxidationby iodine is carried out, and the apparatuses for carrying out suchsynthesis are sold by various sellers including Applied Biosystems. Suchsynthesis of oligonucleotides or analogues thereof may be carried outwithin the ordinary skill of routine workers. Alternatively, antisenseoligonucleotides may be obtained from a number of companies which arespecialized in the syntheses of oligonucleotides. Similar techniques forpreparing other oligonucleotides such as phosphorothioate derivativesand alkylated derivatives are also well-known. Further, for thesyntheses of other modified oligonucleotides such asfluorescence-labeled, biotinylated or cholesterol-modifiedoligonucleotides, use of similar techniques and use of commerciallyavailable modified amidite and CPG product such as biotin, fluorescein,acridine or psoralen-modified amidite and/or controlled porous glass(CPG) (Glen Research, Sterling Va.) is also well-known.

Since antisense method targets the causative gene per se of the diseaseor the disease-related gene per se, it is close to causal therapy. Thus,antisense oligonucleotides have a great potential as therapeutic agentsfor various diseases. For example, U.S. Pat. No. 5,135,917 provides anantisense oligonucleotide which inhibits the expression of humaninterleukin-1 receptor. U.S. Pat. No. 5,098,890 relates to an antisenseoligonucleotide complementary to c-myb oncogene and to an antisenseoligonucleotide therapy of a type of cancerous state. U.S. Pat. No.5,087,617 provides a method for treating cancer patients with anantisense oligonucleotide. U.S. Pat. No. 5,166,195 provides anoligonucleotide inhibitor against HIV. U.S. Pat. No. 5,004,810 providesan oligomer which can hybridize with mRNA of herpes simplex virus Vmw65to inhibit replication thereof. U.S. Pat. No. 5,194,428 provides anantisense oligonucleotide having anti-viral activity against influenzavirus. U.S. Pat. No. 4,806,463 provides an antisense oligonucleotide anduse thereof to the inhibition of replication of HTLV-III. U.S. Pat. No.5,286,717 relates to a mixed binding oligonucleotide phosphorothioatecomplementary to an oncogene. U.S. Pat. Nos. 5,276,019 and 5,264,423relate to phosphorothioate oligonucleotide analogues used for theinhibition of replication of foreign nucleic acids. Effectiveness ofphosphorothioate oligonucleotides against retinitis by cytomegalovirusin AIDS patients have been shown (Bioworld Today, 1994). Thus, it hasbeen established that oligonucleotides are useful means for therapies,and are useful for the treatment of cells and animals (especiallyhuman). Inhibition of expression of casein kinase 2 is also very usefulfor the therapies of kidney diseases.

Since the cDNA sequences of casein kinase 2 are known (for example, thecDNA sequence of human casein kinase 2 α subunit is described in GenBankAccession No. J02853, the cDNA sequence of human casein kinase 2α′subunit is described in GenBank Accession No. M55268, the cDNA sequenceof human casein kinase 2β, subunit is described in GenBank Accession No.AY113186, the cDNA sequence of rat casein kinase 2β subunit is describedin GenBank Accession No. NM_(—)031021, the cDNA sequence of rat caseinkinase 2α subunit is described in GenBank Accession No. L15618, the cDNAsequence of mouse casein kinase 2α subunit is described in GenBankAccession No. AJ001420, and the cDNA sequence of mouse casein kinase 2β,subunit is described in GenBank Accession No. NM_(—)009975. Among these,the cDNA sequences of human casein kinase 2α subunit, α′ subunit and βsubunit, and rat casein kinase 2α subunit and β subunit are shown in SEQID NOs.: 15 to 19, respectively), antisense oligonucleotides against themRNA transcribed from the casein kinase 2 gene may easily besynthesized.

That is, oligonucleotides having nucleotide sequences complementary tothese cDNA sequences may be used as antisense oligonucleotides inprinciple. However, it is preferred to confirm by BLAST search or thelike that the selected nucleotide sequence is specific to the targetgene (i.e., the nucleotide sequence or a nucleotide sequence having ahigh homology thereto does not exist in the genes other than the targetgene). For example, as an antisense oligonucleotide against rat caseinkinase 2 α subunit (include α′ subunit), 5′-gtaatcatcttgattacccca-3′(SEQ ID NO: 1) may be used, and as an antisense oligonucleotide againstrat casein kinase 2β subunit, 5′-ggttggccggccgcttgggcc-3′ (SEQ ID NO: 2)may be used. In addition, oligonucleotides having nucleotide sequencesshown in SEQ ID NOs: 3 to 10 are also exemplified. Further, variousoligonucleotides which hybridize with the mRNA of casein kinase 2 areknown (e.g., WO 020/62951 and WO/020/62954), and these knownoligonucleotides may be used. Nucleotide sequences and parts thereof, aswell as analogues thereof, which correspond beyond the species are alsouseful in the present invention. Not only the coding region of the mRNA,but also other regions such as 5′ untranslated region, 3′ untranslatedregion, 5′ cap and intron/exon junctions, may be employed as the targetregion. That is, the oligonucleotide or its analogue prepared inaccordance with the present invention may target whole or a part ofthese regions, similar to the coding region. In a preferred mode, theoligonucleotide or its analogue may specifically hybridize with thetranscription initiation site, translation initiation site, or withintron/exon junction sites or nucleotide sequence in the 3′ untranslatedregion. The functions of the mRNA to be interfered include all of thefunctions for the survival, such as translocation of mRNA to the site ofprotein translation, actual translation of the protein from mRNA,splicing and maturation of mRNA and independent enzyme activity which ispresumably carried out by mRNA. The overall effect of such aninterference of the functions of the mRNA is to hinder the expression ofthe casein kinase 2 protein. Thus, the antisense oligonucleotide or itsanalogue is the oligonucleotide or its analogue which can at leasthybridize with the mRNA coding for the casein kinase 2 proteinnucleotide sequence-specifically and which can selectively modulate theexpression of casein kinase 2. By administering the antisenseoligonucleotide or its analogue to the cells or individual, expressionof casein kinase 2 alone may be selectively inhibited nucleotidesequence-specifically, so that the kidney diseases may be cured. Theantisense oligonucleotide against casein kinase 2 used in the presentinvention is an oligonucleotide which has a complementarity and canhybridize with the coding region, 3′ untranslated region, 5′untranslated region, 5′ cap and/or intron/exon junctions of the mRNAcoding for casein kinase 2. By using an oligonucleotide havingcomplementarity to the above-described region, it selectively hybridizeswith casein kinase 2. Preferably, the oligonucleotide is anS-oligonucleotide in which at least one binding group in the nucleotideis a sulfur-containing group or phosphorothioate moiety. Pharmaceuticalcompositions containing an effective amount of at least one antisenseoligonucleotide in combination with a pharmaceutically acceptablecarrier are also within the scope of the present invention. Byadministering an antisense oligonucleotide of casein kinase 2,expression of casein kinase 2 may be suppressed, so that kidney diseasesmay be cured. Especially, it is effective for the diseases characterizedby increase of expression of casein kinase 2, such as nephritis. Anexample is described in detail in the Examples below.

RNAi (RNA interference) method is the method employing the phenomenonthat double-stranded RNA administered to cells or individual inhibitsthe expression of the gene (synthesis of protein) having the samesequence. Since RNAi method selectively suppresses the expression of thetarget gene by destroying the target gene or mRNA, it attracts attentionas a method effective for analysis of function of the gene. Further,since RNAi method targets the causative gene per se of the disease orthe disease-related gene per se, it also attracts attention as a methodclose to causal therapy. Double-stranded RNAs having 21-23 base unitsused in RNAi are also called siRNA. siRNAs may easily be designed. Forexample, to avoid the binding site of transcription factor, the firstAA, CA, GA or TA is found from the site downstream of the start codon inthe translated region by not less than 50 bases, and the 21 to 23 basesincluding the subsequent 19 to 21 bases is selected. The selectednucleotide sequence preferably has a GC contents of 30 to 70%, morepreferably 50%, and preferably does not contain GGG or CCC. Then it isconfirmed that the selected nucleotide sequence is specific to thetarget gene by BLAST search or the like. Since an siRNA is also a longchain composed of four types of base units, the siRNA against any targetgene may be simply and routinely synthesized. By virtue of recentprogress, use of siRNA as a novel type of therapeutic method can bedevised now. More particularly, as siRNAs for human casein kinase 2βunit, for example, 5′-cuaccgacaagcucuagactt-3′ and5′-gucuagagcuugucgguagtt-3′ may be used. Alternatively, for example,5′-cuaccgacaagcucuagacat-3′ and 5′-gucuagagcuugucgguagtg-3′ may be used.For rat, 5′-aucuuacuggacucaaugatt-3′ and 5′-gauggcuguucgagaucugtt-3′,for example, may be used. Alternatively, it is preferred to use anoptional similar oligonucleotide or its analogue, which may be preparedby those skilled in the art based on the knowledge of siRNA that ispreferred for the inhibition of expression of casein kinase 2, maypreferably be used. By administering such an siRNA to cells orindividual, expression of casein kinase 2 alone may be selectivelyinhibited nucleotide sequence-specifically, so that the kidney diseasesmay be cured. Especially, it is effective for the diseases characterizedby increase of expression of casein kinase 2, such as nephritis.

These nucleic acids inhibiting casein kinase 2 may be formulated into apharmaceutical composition. The pharmaceutical composition may contain acarrier, thickener, vehicle, buffer, preservative, surfactant, liposomeor lipid formulation, and may also contain one or more activeingredients such as antibiotics and anesthetic drugs. In addition tosuch a pharmaceutical carrier, a cationic liquid for easy incorporationof the oligonucleotide, and/or a lipophilic cationic compound which canform liposomes may be contained in the prescription. One of suchcompositions which attains easy incorporation is lipofectin (BRL,Bethesda Md.). Using liposomes for introducing oligonucleotides intocells is described in, for example, U.S. Pat. Nos. 4,897,355 and4,394,448. Further, for general methods for preparing liposomesconsisting of biological substances, see U.S. Pat. Nos. 4,235,871,4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411 and 4,814,270.Alternatively, the oligonucleotide may be combined with a lipophiliccarrier such as one of a number of sterols including cholesterol, cholicacid and deoxycholic acid. A preferred sterol is cholesterol. Further,the oligonucleotide may be bound to a peptide to be taken by the cells.Examples of the effective peptides include peptide hormones, antigens,antibodies and peptide toxins. By choosing a peptide selectively takenby the cells constituting kidney, specific delivery of theoligonucleotide may be efficiently carried out. The oligonucleotide maybe bound via covalent bond through 5′ OH group by the formation of anactivated aminoalkyl derivative. The selected peptide may then be boundvia covalent bond to the oligonucleotide activated by using an amino-and sulfhydryl-reactive hetero bifunctional reagent. The latter binds tocystein residues existing in the peptide. By exposing the cells to theoligonucleotide bound to the peptide, the peptidyl antisense reagent issubjected to endocytosis, and the oligonucleotide binds to the targetmRNA to suppress translation. See PCT/US89/02363. The administrationdose depends on the severity of the state to be treated and sensitivity,and the treatment is continued for several days to several months untilthe cure is attained or regression of the diseased state is attained.Optimum administration scheme may be calculated based on the measurementof the drug accumulated in the body. Those skilled in the art may easilydetermine the optimum dose, administration method and repetitionfrequency. Although the optimum dose may vary depending on the relativeeffectivity of each oligonucleotide, it may be calculated, in general,based on EC50 determined by animal experiments. For example, once themolecular weight of the compound (determined by the oligonucleotidesequence and chemical structure) and effective dose such as IC50(experimentally determined) are given, the administration dose in termsof mg/kg is routinely calculated. A number of references relate toadministration of genes to mammals. Transfer of genes via liposomes anddirect transfer of genes to tissues are described in, for example,Brigham et al., (1989) Am. J. Med. Sci., 298:278-281; Nabel et al.,(1990) Science, 249:1285-1288; Hajinski et al., (1991) Am. J. Resp. CellMolec. Biol., 4:206-209; Wang and Huang (1987), Proc. Natl. Acad. Sci.,USA., 84:7815-7855. An example of review of human gene therapy isAnderson, Science (1992) 256:808-813.

Although the administration dose of the oligonucleotide is appropriatelyselected as described above, in most cases, a dose of about 0.005 mg/kgto 5 mg/kg per day in terms of the weight of the oligonucleotide isappropriate. The administration route is preferably parenteraladministration such as intravenous administration, intramuscularadministration or subcutaneous administration.

Among the substances which inhibit casein kinase 2, as the substanceswhich inhibit the enzyme activity, anti-casein kinase 2 antibodies andthe antigen-binding fragments thereof, which at least partiallyneutralize the activity of casein kinase 2 may be used. The anti-caseinkinase 2 antibody is a monoclonal antibody and/or polyclonal antibody ofwhich corresponding antigen is a subunit of casein kinase 2 or a partialpeptide of casein kinase 2 protein, or antigen-binding fragment thereof,and is not restricted as long as it can neutralize the casein kinase 2activity at least partially. They may be prepared by a conventionalmethod using casein kinase 2 or its subunit as an antigen. A polyclonalantibody having neutralizing activity may be recovered from an antiserumobtained by using casein kinase 2 as an immunogen by a conventionalmethod. A monoclonal antibody having neutralizing activity may beobtained by preparing monoclonal antibodies by a conventional methodusing casein kinase 2 or its subunit as an antigen, and screening themonoclonal antibody having neutralizing activity of casein kinase 2. Theanti-casein kinase 2 antibody may be bound to a peptide to be taken bythe cells. As mentioned above, examples of the effective peptidesinclude peptide hormones, antigens, antibodies and peptide toxins. Bychoosing a peptide selectively taken by the cells constituting kidney,specific delivery of the anti-casein kinase 2 antibody may beefficiently carried out. By administering such an anti-casein kinase 2antibody to cells or individual, casein kinase 2 activity alone may beselectively inhibited, so that the kidney diseases may be cured.Especially, it is effective for the diseases characterized by increaseof expression of casein kinase 2, such as nephritis.

Preferred examples of the substance which inhibits the enzyme activity,amongst the substances that inhibit casein kinase 2, include thecompounds having the structure shown by Formula 1 below.

(wherein R¹ and R² independently represent hydrogen, hydroxy or phenylwhich may have one or more substituents selected from those representedby Formula 2, with the proviso that at least one of R¹ and R² is saidphenyl group which may have said substituent(s); R³ and R⁴ independentlyrepresent hydrogen or a substituent represented by Formula 2; and R⁵represents hydrogen, sugar residue, C₁-C₄ alkyl or acyl);—OR⁵  Formula 2(wherein R⁵ represents the same meanings as R⁵ in Formula 2).

In Formula 1, preferred examples of the sugar residue include glucose,galactose and ribose. Preferred examples of C₁-C₄ alkyl includes methyland ethyl. Preferred examples of the acyl include C₁-C₄ acyl such asacetyl, and benzoyl.

Preferred examples of the substance which inhibits the enzyme activity,amongst the substances that inhibit casein kinase 2, further include thefollowing compounds:

(wherein R⁶ and R⁷ independently represent hydrogen, halogen or C₁-C₄alkyl); TBB (Sarno S, FEBS Letters 2001 469:44-48) which is4,5,6,7-tetrabromobenzimidazole; DRB (Meggio F, Eur. J. Biochem., 1990187:89-94) which is 5,6-dibromo-1-(β-D-ribofuranosyl)benzimidazole;compounds having the structures of:

(wherein R⁸ represents phenyl which may be substituted by 1 to 3substituents selected from halogens, methyl, trifluoromethyl andhydroxy, or quinolyl, ethylmethyl sulfide, t-butylphenylthio orN-ethylphenylamino; and R⁹ represents hydroxy or amino);

(wherein n represents 1 to 3; X represents —NH— or —CONH—; and Yrepresents hydroxy, piperidine, pyrrolidine, piperazine, pyrazole ortriazole);

(wherein R¹⁰ represents 2-thienyl or phenyl which may be substituted byhalogen, methyl, ethyl or t-butyl; and R¹¹ represents —SO₂—NH₂,—SO₂—NHMe or —SO₂—NH[(CH₂)₂N(CH₃)₂].

A preferred example of the compounds represented by the above-describedformulae is apigenin represented by Formula 7 below. Apigenin inhibitsor decrease the functional activity of casein kinase 2 by about 25% at aconcentration of 10 μM, and by about 75% at a concentration of 100 μM,so that it is preferred.

Further, especially preferred examples of the compounds represented bythe above-described formulae include the following compounds:

R1 R2

—OH

—OH

—OH

—OH

—NH2

—NH2 —CH₂CH₂SMe —NH2

—OH

—OH

R —CONHCH₂CH₂CH₂OH —NHCH₂CH₂CH₂-(1-piperidine) —NHCH₂CH₂CH₂-(1-pyrazole)—NHCH₂CH₂CH₂-(1,2,4-triazole)

R1 R2 2-thienyl 4-SO₂—NH₂ (3-Cl)-2-thienyl 4-SO₂—NH₂ 3-thienyl 4-SO₂—NH₂(5-Et)-2-thienyl 4-SO₂—NH₂ 5-[C(CH₃)₃]-2-thienyl 4-SO₂—NH₂(5-Et)-2-thienyl 4-SO₂—NHMe (5-Et)-2-thienyl 4-SO₂—NH[(CH₂)₂N(CH₃)₂]

These compounds per se, the processes for producing the compounds, andthe facts that the compounds have inhibitory effects against the enzymeactivity of casein kinase 2 are known, and described in, for example,U.S. Pat. No. 6,358,978, PCT publication Nos. WO 01/42231, WO 02/24681and WO 02/057240. Therefore, these compounds may easily be produced byknown methods.

The compounds which inhibit enzyme activity of casein kinase 2 are notrestricted to the compounds described above, and other compounds knownto inhibit enzyme activity of casein kinase 2 may also be used.

The above-described various substances which inhibit casein kinase 2 maybe in the forms of pharmaceutically acceptable salts. These arewell-known bases or acid addition salts. Examples of basic salts includethose derived from ammonium hydroxide, alkaline and alkaline earth metalhydroxides, carbonates and hydrogen carbonates; and those derived fromaliphatic and aromatic amines, aliphatic diamines andhydroxyalkylamines. Examples of the base especially useful forpreparation of these salts include ammonium hydroxide, potassiumcarbonate, sodium hydrogen carbonate, lithium hydroxide, calciumhydroxide, methylamine, diethylamine, ethylenediamine, cyclohexylamineand ethanolamine. Potassium salt, sodium salt and lithium salt areespecially preferred. As the acid addition salts, addition salts formedwith pharmaceutically acceptable non-toxic acids including inorganicacids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid and phosphoric acid; and organic acids including organic carboxylicacids such as glycolic acid, maleic acid, fumaric acid, malic acid,tartaric acid, citric acid, salicylic acid and o-acetoxybenzoic acid,and organic sulfonic acids such as methanesulfonic acid,2-hydroxyethanesulfonic acid, p-toluenesulfonic acid andnaphthalen-2-sulfonic acid, are preferred.

These compounds which inhibit enzyme activity of casein kinase 2 may beused individually, or two or more of these compounds may be used incombination.

When the agent for therapy and/or prevention of kidney diseasesaccording to the present invention, comprising as an effectiveingredient the compound which inhibits the enzyme activity of caseinkinase 2 is administered to an animal or human, it may be administeredthrough any administration route such as oral, intravenous,intramuscular or subcutaneous administration, or by directadministration into kidney tissue. The agent may be formulated into onesuitable for the respective administration method. Examples of theformulation of the agent for therapy and/or prevention of kidneydiseases according to the present invention include injections,sublingual tablets, percutaneous cataplasms, tablets, capsules, subtlegranules, granules, powders, balls, troches, syrups, emulsions,suspensions, suppositories, ointments and instillations. Depending onthe formulation, a pharmaceutically acceptable vehicle, such as lactose,potato starch, calcium carbonate or sodium alginate may be added.Further, other materials which are usually used for formulation, forexample, proteins such as serum albumin, salts for buffer action or foradjusting osmosis, carriers, vehicles, bases, solubilizing agents,dispersants, stabilizers, antioxidants, preservers, disintegrators,binders, corrigents, lubricants, coating agents, coloring agents and thelike may be blended.

Although the administration dose may be widely varied depending on theadministration method, the type of the warm-blooded animal includinghuman to be treated, age of the individual, body weight, severity of thesymptom, and on the diagnosis by the physician, the dose is usuallywithin the range of 0.001 to 100 mg/kg body weight per day. However, thedose range may be changed depending on the severity of the symptom ofthe patient and on the diagnosis of the physician. The above-mentioneddose may be administered once a day or dividedly several times per day.In case of the above-mentioned gene therapy, it may be carried outreferring to the guideline by NIH, including the judgment of safety(Recombinant DNA Advisory Committee, Human Gene Therapy, 4,365-389(1993)). The agent for therapy and/or prevention of kidneydiseases according to the present invention may be used literally forthe purpose of therapy and/or prevention of kidney diseases, and it mayalso be administered for the purpose of keeping (prevention ofexacerbation), alleviation (amelioration of symptom). Further, thepresent invention includes therapeutic methods of kidney diseases usinga compound which inhibits casein kinase 2.

The agent for therapy and/or prevention of kidney diseases according tothe present invention may be used in combination with one or more drugssuch as steroids, immunosuppressants, antihypertensive drugs (e.g.,calcium blockers, angiotensin converting enzyme inhibitors, ATIIreceptor blockers and α receptor blocking drugs), antiplatelet drugs(e.g., dipyridamole and dilazep), and anticoagulants. Further, it may beused in combination with gene therapy for introducing HGF or the like.When using these drugs in combination, each drug may be formulatedseparately or in combination, by mixing with one or morepharmaceutically acceptable carriers, vehicles, binders, diluents andthe like, and the resulting pharmaceutical composition may beadministered orally or parenterally. In cases where the drugs areseparately formulated, the separate formulations may be mixed using adiluent or the like when use and the mixture may be administered, or theseparate formulations may be administered to the same subjectsimultaneously or with a time interval.

As mentioned above, the present inventors discovered that the expressionamount of casein kinase 2 gene in the kidney cells of diseased kidneywas significantly larger than the expression amount of normal kidneycells. Therefore, the present invention also provides a method fordiagnosis of kidney diseases, comprising measuring activity and/orcontent of casein kinase 2, and/or measuring expression amount of caseinkinase 2 gene in a sample separated from body. Here, as the “sampleseparated from body”, cells constituting kidney are preferred.

The diagnostic method of kidney diseases employing the expression ofcasein kinase 2 as an index may be carried out by using as an index theexpression of α subunit, α′ subunit and/or β subunit of casein kinase 2,particularly expression of the gene, protein and/or the enzyme activity.For example, in cases where the expression of the gene or protein isused as the index, firstly, RNAs and proteins are extracted from thekidney to be subjected to diagnosis and from a kidney of non-diseasedstate used as a control. Using a prescribed amount of the extracted RNAsor proteins, expression of casein kinase 2 is detected, respectively. Bycomparing the expression of casein kinase 2 in the kidney to bediagnosed and the expression in the non-diseased kidney used as acontrol (healthy individual), the state of the kidney disease in thekidney to be subjected to diagnosis may be diagnosed. If the expressionof casein kinase 2 in the kidney subjected to diagnosis is higher thanthat in the control non-diseased kidney, the kidney subjected todiagnosis is diagnosed as in the diseased condition, especially in thediseased condition involving casein kinase 2. By measuring theexpression of casein kinase 2 in the tissue or cells of kidney usingsuch a detection system, the severity of the kidney disease may bemeasured accurately.

Expression of the casein kinase 2 gene may be measured by using RT-PCRmethod (Polymerase chain reaction method) (“PCR Protocols” Innis M A,Gelfad D H, Sninsky J J and White T J eds., Academic Press, Sandiego(1990)), Northern blot method (Molecular Cloning, Cold Spring HarborLab. (1989)), array method, DNA chip method, in situ hybridizationmethod, in situ RT-PCR (Nucl. Acids Res., 21, 3159-3166(1993)) or thelike. Since any of these methods per se is well-known, and since thecDNA sequence of casein kinase 2 is also known as mentioned above, thoseskilled in the art may easily measure the expression of casein kinase 2gene in the cells, and an example is described in detail in Examplesbelow. Measurement by RT-PCR is preferably employed. That is, from aprescribed amount of the extracted RNAs, cDNAs are synthesized using areverse transcriptase. By using a prescribed amount of each synthesizedcDNA as a template, PCR is performed using primers for casein kinase 2,thereby amplifying the cDNA. A prescribed amount of each PCR product issubjected to agarose gel electrophoresis, and then ethidium bromidestaining is performed to detect expression of casein kinase 2 gene. WhenPCR is performed in relation to the gene of casein kinase 2, it ispreferred to carry out PCR in the similar manner using a constantlyexpressing gene (e.g., a house keeping gene such as G3PDH or β actin) asan internal standard. After subjecting a prescribed amount of the PCRproduct to agarose gel electrophoresis, ethidium bromide staining isperformed to detect the expression of the gene, and the result of thedetection of the casein kinase 2 gene is compensated. Any of these stepsare conventional. It is preferred to sequence each of the PCR productsby a conventional method such as dideoxy method (Proc. Natl. Acad. Sci.,USA., 74, 5463(1977)) or Maxam Gilbert method (Methods in Enzymology,65, 499(1980)), or simply by using a commercially available sequence kitor the like. The primers used for the PCR of casein kinase 2 gene arenot restricted as long as they can specifically amplify the gene of eachsubunit of casein kinase 2, and they may be appropriately designed basedon the known nucleotide sequences (supra) of the full length cDNAs ofthese genes. That is, as the sense primer, an oligonucleotide having anucleotide sequence which is the same as that of the sense chain of cDNAof casein kinase 2 may be employed, and as the antisense primer, anoligonucleotide having a nucleotide sequence which is the same as thatof the antisense chain of cDNA of casein kinase 2 may be employed. Thearea sandwiched between the sense primer and the antisense primer isamplified by PCR. The oligonucleotides used as primers may besynthesized by a conventional method using a commercially availablechemical synthesizer. The size of the primer is not restricted, andusually about 15 to 30 bases. For example, in case of β subunit of ratcasein kinase 2,5′-ccgcggacataaagatgagt-3′ (SEQ ID NO: 11) may be usedas the sense primer, and 5′-aaaccagtgccgaagtatgc-3′ (SEQ ID NO: 12) maybe used as the antisense primer. In case of α subunit of rat caseinkinase 2,5′-agaaagcttcggctaataga-3′ (SEQ ID NO: 13) may be used as thesense primer, and 5′-actgaagaaatccctgacat-3′ (SEQ ID NO: 14) may be usedas the antisense primer. By this, expression of the casein kinase 2 genemay be detected specifically and simply. Alternatively, quantitative PCRsuch as the so called realtime-detection PCR using a quencherfluorescent dye and a reporter fluorescent dye may also be employed.Since the realtime-detection PCR is also well-known, and kits thereforare commercially available, it may be easily carried out.

The oligonucleotide according to the present invention may also be usedas a probe, and is useful for the detection of the expression of caseinkinase 2 gene and for the diagnosis. For example, it may be used as theprobe used in the above-described Northern blot method, array method,DNA chip method, in situ hybridization method and the like. Thesemethods per se are well-known, and those skilled in the art can easilycarry out them. A labeled probe obtained by labeling the above-describedoligonucleotide with a label such as fluorescent label, radioactivelabel or biotin label may be used. Whether the test nucleic acid existsin the sample or not may be determined by immobilizing the test nucleicacid or its amplification product on a solid phase, hybridizing the testnucleic acid with the labeled probe, and measuring the label bound tothe solid phase after washing. Alternatively, the test nucleic acid maybe detected by immobilizing a nucleic acid for measurement on a solidphase, hybridizing the test nucleic acid with the immobilized nucleicacid for measurement, and detecting the test nucleic acid bound to thesolid phase with the labeled probe or the like. In such a case, thenucleic acid for measurement immobilized on the solid phase is alsocalled probe. Methods for measuring a test nucleic acid using a nucleicacid probe are also well-known in this field, and may be carried out bycontacting the nucleic acid probe with the test nucleic acid in a bufferat Tm or vicinity thereof (preferably within ±4° C.) to hybridize them,and after washing, by measuring the hybridized labeled probe or thetemplate nucleic acid bound to the solid phase probe. Such methodsinclude well-known methods such as the above-mentioned Northern blot, insitu hybridization and Southern blot method.

For example, an oligonucleotide labeled with a radioactive label may beprepared by labeling the 5′ end thereof with ³²P by polynucleotidekinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989, Vol. 2, p10. 59. Then theoligonucleotide labeled with the radioactive label is made to contactwith the tissue or cell sample, and then the sample is washed to removethe unbound oligonucleotide. The radioactivity remaining in the sampleindicates the bound oligonucleotide (it indicates the expression ofcasein kinase 2), and the radioactivity may be measured by ascintillation counter or other ordinary means. By performingautoradiography of the tissue using the radioactive labeledoligonucleotide, localization, distribution and/or the amount of theexpression of casein kinase 2 gene may be determined. Alternatively,thin section of the tissue may be treated with the radioactive labeledoligonucleotide, and then exposed to photograph emulsion in accordancewith the routine autoradiography, after the washing as mentioned above.Upon development, the emulsion gives an image of silver particles in thearea in which casein kinase 2 gene is expressed. By quantification ofthe silver particles, expression of casein kinase 2 may be detected. Asimilar assay for the detection of casein kinase 2 gene expression bydetection of fluorescence may be developed using the oligonucleotideaccording to the present invention conjugated with fluorescein or otherfluorescent label in place of the radioactive label. Each of these modesis known in this field. Those skilled in the art can easily apply theseknown modes to the detection of expression of casein kinase 2 geneaccording to the technique of the present invention, thereby novel anduseful means for detecting the expression of casein kinase 2 areprovided.

In cases where expression of the casein kinase 2 protein is used as anindex, casein kinase 2 protein or its partial peptide is detected usinga monoclonal antibody and/or polyclonal antibody of which correspondingantigen is a subunit of casein kinase 2 or a partial peptide of caseinkinase 2 protein, or antigen-binding fragment thereof, by enzyme-linkedimmunosorbent assay (ELISA), radio immunoassay (RIA), enzyme immunoassay(EIA), Western blot method, dot blot method, protein chip analysismethod, immunostaining analysis method or the like. The antibody or apart thereof used for the detection of casein kinase 2 protein is notrestricted as long as it can specifically detect the subunit of caseinkinase 2 or a partial peptide of casein kinase 2 protein alone. Thepolyclonal antibody may be obtained by administering the antigens ofcasein kinase 2 protein to a rabbit, rat, mouse, goat or the likethrough parenteral administration, such as intravenous, intramuscular orsubcutaneous administration. The monoclonal antibody may be obtained byan ordinary method which is performed by, for example, removing spleencells of a mouse to which the antigens of casein kinase 2 protein wereorallyparenterally administered, fusing the spleen cells with mousemyeloma cells, and purifying the culture supernatant of the fused cells.Casein kinase 2 per se may also be prepared easily by a known method.For example, casein kinase 2 may be obtained by extracting casein kinase2 from an organ such as kidney, liver, spleen, lung, bone marrow, brainor placenta, or from blood cells, of a mammal such as rat or bovine.Alternatively, casein kinase 2 may be obtained by culturing primarycultured cells or established cell line producing casein kinase 2, andseparating and purifying casein kinase 2 from the cells. Alternatively,in accordance with a known genetic engineering technique, the genecoding for casein kinase 2 is incorporated into appropriate host cellssuch as cells of E. coli, Bacillus subtilis, yeasts, filamentousbacteria, plants and animals, and the desired recombinant casein kinase2 may be obtained from the culture of the transformants, which may becarried out easily. The above-mentioned various immunoassays and otheranalysis methods per se are well-known. For example, the enzyme-linkedimmunosorbent assay may be carried out by placing a test sample in apolystyrene microplate on which an anti-casein kinase 2 antibody isimmobilized, washing the plate after reaction for a prescribed time,adding an anti-casein kinase 2 antibody labeled with an enzyme such asperoxidase, washing the plate again, and adding a substrate such ashydrogen peroxide and a coloring agent such as OPD (o-phenylenediamine)to allow coloring. The method for detecting casein kinase 2 in thetissue by the immunostaining analysis method may be carried out bystaining a removedisolated tissue with an anti-casein kinase 2 antibodylabeled with a fluorescent substance such as FITC (fluorescenceisothiocyanate) or with an enzyme label such as peroxidase. According tothe present invention, by detecting casein kinase 2 using such adetection system, the severity of the kidney disease may be measured,and this may be carried out easily.

In cases where the expression of enzyme activity of casein kinase 2 isused as the index, the enzyme activity of casein kinase 2 protein orpartial polypeptide thereof when the enzyme substrate is made to contactwith the casein kinase 2 protein or partial polypeptide thereof isdetected, by using protein phosphorylation analysis method,intracellular localization change analysis method or enzyme activityanalysis method. In these methods, as the enzyme substrate, anysubstance which can serve as an enzyme substrate of casein kinase 2 orthe peptide, and usually, casein protein or a partial peptide of caseinkinase 2 protein is used. Further, as mentioned above, casein kinase 2may be obtained by various methods. For example, casein kinase 2 may beobtained by extraction and purification from organs of mammals or byseparation and purification from cell culture. Alternatively,recombinant casein kinase 2 may be obtained from the culture of thetransformants prepared by incorporating the gene coding for caseinkinase 2 into appropriate host cells by the known genetic engineeringmethod. The thus obtained casein kinase 2 may be used for confirming theinhibitory activity of the substance which inhibits the enzyme activityof casein kinase 2, even if a part of the amino acid sequence thereof isdeleted or substituted, or other amino acid sequence is inserted, aslong as it has the enzyme activity. In a method for analyzing the enzymeactivity, for example, a sample (10 ng to 0.1 mg protein) is incubatedwith a peptide Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu orArg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu (0.2 mM) as the enzyme substrate ina buffer (20 mM MOPS, pH 7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1mM sodium o-vanadate, 1 mM dithiothreitol, 15 mM MgCl₂, 0.1 mM [γ-³²P]GTP or [γ-³²P]ATP, 0.002 mCi) at 37° C. for 10 minutes. After theincubation, 40% trichloroacetic acid is added to stop the reaction, andan aliquot of the reaction solution is taken on phosphocellulose paper(1 to 1.5 cm square, Whatman P81 or the like). The phosphorylation ofthe peptide which is the enzyme substrate may be measured by washing thephosphocellulose paper with 0.75% phosphoric acid 3 times for 5 minutesper wash, then further washing the paper with acetone for 5 minutes, andmeasuring the radioactivity of ³²P on the phosphocellulose paper by ascintillation (Econofluor-2, NEN or the like) (R. Roskoski, MethodsEnzymol. Vol. 99, pp. 3-6, (1983)). High radioactivity of ³²P measuredhere indicates high expression of the enzyme activity of the caseinkinase 2 protein or a partial peptide thereof in the sample. The enzymeactivity analysis method similar to this method may be carried outeasily because a commercially available enzyme activity-measuring kit(Casein kinase 2 Kinase Assay, Upstate) may be used.

Techniques for the production and purification of the above-describedprimers and antibodies are well-known in this field. The method fordiagnosis of kidney diseases utilizing the expression of casein kinase 2according to the present invention includes, as mentioned above, themethods detecting expression of casein kinase 2 gene, expression of theprotein and expression of the enzyme activity. To detect the geneexpression of casein kinase 2, casein kinase 2 gene, primers and/orprobe thereto may be used. To detect casein kinase 2, the antibody or apart thereof, to casein kinase 2 or a partial peptide thereof, or anucleic acid (aptamer) which specifically recognizes casein kinase 2 maybe used. Further, use of an enzyme substrate of casein kinase 2 for thediagnosis of kidney diseases is included. These techniques may beappropriately selected, and by using a reagent kit for the detection ofthe expression of casein kinase 2, diagnosis of kidney diseases may becarried out simply. Thus, in accordance with the diagnostic method ofkidney diseases according to the present invention, diagnostic reagentkits and diagnostic reagents of kidney diseases are provided.

The method for diagnosis of kidney diseases according to the presentinvention may be not only applied widely to the diagnoses of humankidney diseases, but also applied to various model animals of kidneydiseases. Especially, for glomerulonephritis, findings suggesting theinvolvement of immunological mechanism have been reported in a number ofcases, and preparation of animal experiment models as models of humanchronic nephritis has been widely studied. The method of the presentinvention may preferably be used for the animal models of kidneydiseases induced by anti-glomerular basement membrane antibody. That is,by the method of the present invention, whether a kidney disease animalmodel really suffers from a kidney disease or not may easily be checked.A kidney disease animal model induced by anti-glomerular basementmembrane antibody may be prepared by administering an anti-glomerularbasement membrane antiserum obtained from rabbit to rat or mouse. Theanti-glomerular basement membrane antiserum may be prepared by preparingglomerular basement membrane from renal cortex of rat or mouse,administering the glomerular basement membrane to rabbit together withFreund's complete adjuvant, and obtaining serum (“Method for ScreeningPharmacological Effect for Development of New Drugs—Latest Trends andFacts—Vol. 1”, supervised by Hikaru OZAWA, Shisei Shoin, 1984, p. 143).The model is widely used as the most common disease model of primaryglomerulonephritis. According to the classification based on morphologyand clinical symptoms, it is the disease model closest tocrescent-forming glomerulonephritis and acute progressiveglomerulonephritis, among the primary glomerulonephritis. The diseasedstate may be grasped by measuring urinary protein excretion which is akidney function marker, blood creatinine level or endogenous creatinineclearance. Since creatinine is not absorbed by kidney at all, if thefiltering function of the glomeruli is deteriorated by a kidney disease,the blood creatinine level is increased. Therefore, blood creatininelevel and creatinine clearance are good markers of kidney function. Inthe second week from the induction of nephritis, the blood creatininelevel is increased twice of the normal value, and the animal present thediseased state of nephritis and renal failure accompanying the generaldecrease in the filtration ability of glomeruli. Such an animal model ofkidney diseases, produced by induction by anti-glomerular basementmembrane antibody, is widely used in the world as the model of humanchronic nephritis. The animal model may be used by the method of YoshioSUZUKI et al. (Journal of Japan Society of Nephrology, Vol. 23, pp.323-331, 1981) and (Journal of Japan Society of Nephrology, Vol. 77, pp.407-417, 1981). The utility of the drug according to the presentinvention may also be confirmed. Other useful animal models of kidneydiseases include 1) those prepared by Thy-1 monoclonal antibody oranti-thymus antibody, 2) hereditary nephrosis rats and mice, 3)spontaneous diabetes models of mice and rats, 4) diabetes models inducedby administering streptozotocin or alloxan to mice and rats, and 5)models from which 5/6 of the kidney was resected.

The present invention will be described more concretely by way ofexamples below.

EXAMPLE 1

The relationship between the expression of casein kinase 2 in the kidneytissue, that is, in the cells constituting the kidney and the state ofthe kidney disease was analyzed. More particularly, using rats, thestate of the kidney disease was diagnosed using the urinary proteinexcretion which is a widely used kidney function marker, and theexpression of casein kinase 2α subunit in the kidney tissue was detectedby RT-PCR method, so as to analyze the relationship between the state ofthe kidney disease and the expression of casein kinase 2.

To rats (Wistar-Kyoto strain, male, body weight 190 to 210 g, CharlesRiver Japan, Inc.), rabbit anti-glomerular basement membrane antibodywas intravenously administered (0.3 ml/kg) to induce nephritis. To therats of normal group, normal rabbit serum (0.3 ml/kg) was intravenouslyadministered. On 7 to 14 days after the induction of nephritis, all ratswere placed in metabolic cages, and urine excreted during 24 hours wascollected and the amount of the urine was measured. The urine was thencentrifuged at 3000 rpm for 15 minutes, and the urine protein in thesupernatant was measured using TP Test Wako (Wako Pure Chemical).Further, after the collection of urine, the kidneys were isolated, andRNAs were extracted therefrom by a conventional method, followed bypreparation of cDNAs from 1 μg of RNAs using a commercially availablereverse transcription reaction kit (Invitrogen). Using 1 μl each of thesynthesized cDNAs as templates, PCR was performed using the primers forcasein kinase 2α subunit and using a commercially available PCR kit(Takara) to amplify the cDNA. Primers were prepared based on thenucleotide sequence of the full length cDNA of rat casein kinase 2αsubunit. That is, as the sense primer, 5′-agaaagcttcggctaataga-3′ wasused, and as the antisense primer, 5′-actgaagaaatccctgacat-3′ was used.The PCR was carried out by repeating 30 times the thermal cycle of 94°C. for 30 seconds, 58° C. for 30 seconds and 72° C. for 40 seconds, andthe reaction was stopped finally at 4° C. Each of 10 μl aliquots of theamplified PCR products was electrophoresed on agarose gel, and the gelwas subjected to ethidium bromide staining, followed by measuring theexpression of the casein kinase 2α subunit gene. This measurement wascarried out by taking photographs of the detected casein kinase 2 so asto make image files of the expression of the casein kinase 2 gene, anddegitalizating the image file using a software NIH Image on Macintosh.When the PCR was carried out, a constantly expressing gene (a housekeeping gene, G3PDH) was detected as an internal standard, and theresults of detection of the expression of casein kinase 2α subunit genewas compensated.

As a result, as shown in FIG. 1, the expression amount of casein kinase2α subunit gene in the kidney tissue, i.e., in the cells constitutingthe kidney in the diseased state, is prominently larger than that in thenormal kidney. Further, it can be easily seen that the larger theexpression amount of the casein kinase 2 gene in the cells constitutingthe kidney, the worse the state of the kidney disease, and the closerthe expression amount to the normal level, the closer the state of thekidney disease to the normal state.

EXAMPLE 2

Using the expression of casein kinase 2α subunit gene as an index,kidney disease was diagnosed. That is, using rats, expression of caseinkinase 2α subunit gene in the kidney tissue, i.e., in the cellsconstituting the kidney, was detected by RT-PCR method, and the kidneydisease was diagnosed using it as an index.

First, to rats (Wistar-Kyoto strain, male, body weight 190 to 210 g,Charles River Japan, Inc.), rabbit anti-glomerular basement membraneantibody was intravenously administered (0.3 ml/kg) to induce nephritis.To the rats of normal group, normal rabbit serum (0.3 ml/kg) wasintravenously administered. After the induction of nephritis, all ratswere placed in metabolic cages, and urine excreted during 24 hours wascollected, and then the kidneys were isolated. RNAs were extracted fromeach kidney to be subjected to diagnosis and from each kidney ofnon-diseased state as a control by a conventional method, and cDNAs weresynthesized from 1 μg of the respective RNAs using a commerciallyavailable reverse transcription reaction kit (Invitrogen). Using 1 μleach of the synthesized cDNAs as templates, PCR was performed using theprimers for casein kinase 2α subunit and using a commercially availablePCR kit (Takara) to amplify the cDNA. Primers were prepared based on thenucleotide sequence of the full length cDNA of rat casein kinase 2αsubunit. That is, as the sense primer, 5′-agaaagcttcggctaataga-3′ wasprepared and used, and as the antisense primer,5′-actgaagaaatccctgacat-3′ was prepared and used. The PCR was carriedout by repeating 30 times the thermal cycle of 94° C. for 30 seconds,58° C. for 30 seconds and 72° C. for 40 seconds, and the reaction wasstopped finally at 4° C. Each of 10 μl aliquots of the amplified PCRproducts was electrophoresed on agarose gel, and the gel was subjectedto ethidium bromide staining, followed by measuring the expression ofthe casein kinase 2 gene. This measurement was carried out by takingphotographs of the detected casein kinase 2 so as to make image files ofthe expression of the casein kinase 2 gene, and degitalizating the imagefile using a software NIH Image on Macintosh. When the PCR was carriedout, a constantly expressing gene (a house keeping gene, G3PDH) wasdetected as an internal standard, and the results of detection of theexpression of casein kinase 2α subunit gene was compensated.

Expression of the casein kinase 2α subunit gene in the kidney subjectedto diagnosis, and the expression of the casein kinase 2α subunit gene inthe non-diseased kidney used as a control were compared. As a result,expression of the casein kinase 2α subunit gene in the kidney subjectedto diagnosis was higher than that in the non-diseased kidney used as anegative control (Table 1). Therefore, the kidney subjected to diagnosiswas diagnosed to be in the diseased condition, especially in thediseased condition involving casein kinase 2. To verify these diagnosisresults, the urinary protein excretion which is a widely used kidneyfunction marker of each rat individual diagnosed as in the diseasedcondition was measured. The urinary protein excretion of eachnon-diseased individual used as a control was also measured. As aresult, the urinary protein amounts of the individuals diagnosed as inthe kidney-diseased state were clearly larger than those of theindividuals of non-diseased state (Table 2). Thus, it was confirmed thatthe individuals diagnosed as in the kidney-diseased state were actuallyin the kidney-diseased state, and it was proved that the diagnosticmethod according to the present invention can accurately judge thediseased state of the kidney disease. TABLE 1 Results of Detection ofExpression of Casein Kinase 2 α Subunit Gene When Kidney Disease wasDiagnosed Using Expression of Casein Kinase 2 α Subunit Gene as Indexare Shown in Terms of Values. Expression Ratio Kidney (casein kinase2/G3PDH) Kidney Subjected to Diagnosis 3.4 Non-diseased Kidney 1.1

TABLE 2 Results of Measurement of Urine Protein of Individuals Diagnosedas in Kidney-diseased State Using Expression of Casein Kinase 2 αSubunit Gene as Index Individual Urine Protein (mg/day) IndividualDiagnosed as in Kidney-diseased State 142.9 Individual Not inKidney-diseased State 28.7

EXAMPLE 3

Using the expression of casein kinase 2β subunit gene as an index,kidney disease was diagnosed. That is, using rats (Wistar-Kyoto strain,male, body weight 190 to 210 g, Charles River Japan, Inc.), expressionof casein kinase 2β subunit gene in the kidney tissue, that is, in thecells constituting the kidney, was detected by RT-PCR method, and thekidney disease was diagnosed using it as an index.

First, to rats, rabbit anti-glomerular basement membrane antibody wasintravenously administered (0.3 ml/kg) to induce nephritis. To the ratsof normal group, normal rabbit serum (0.3 ml/kg) was intravenouslyadministered. After the induction of nephritis, all rats were placed inmetabolic cages, and urine excreted during 24 hours was collected, andthen the kidneys were isolated. RNAs were extracted from each kidney tobe subjected to diagnosis and from each kidney of non-diseased state asa control by a conventional method, and cDNAs were synthesized from 1 μgof the respective RNAs using a commercially available reversetranscription reaction kit (Invitrogen). Using 1 μl each of thesynthesized cDNAs as templates, PCR was performed using the primers forcasein kinase 2β subunit and using a commercially available PCR kit(Takara) to amplify the cDNA. Primers were prepared based on thenucleotide sequence of the full length cDNA of rat casein kinase 2βsubunit. That is, as the sense primer, 5′-ccgcggacataaagatgagt-3′ wasprepared and used, and as the antisense primer,5′-aaaccagtgccgaagtatgc-3′ was prepared and used. The PCR was carriedout by repeating 30 times the thermal cycle of 94° C. for 30 seconds,58° C. for 30 seconds and 72° C. for 40 seconds, and the reaction wasstopped finally at 4° C. Each of 10 μl aliquots of the amplified PCRproducts was electrophoresed on agarose gel, and the gel was subjectedto ethidium bromide staining, followed by measuring the expression ofthe casein kinase 2 gene. This measurement was carried out by takingphotographs of the detected casein kinase 2 so as to make image files ofthe expression of the casein kinase 2 gene, and degitalizating the imagefile using a software NIH Image on Macintosh. When the PCR was carriedout, a constantly expressing gene (a house keeping gene, G3PDH) wasdetected as an internal standard, and the results of detection of theexpression of casein kinase 2β subunit gene was compensated.

Expression of the casein kinase 2β subunit gene in the kidney subjectedto diagnosis, and the expression of the casein kinase 2β subunit gene inthe non-diseased kidney used as a control were compared. As a result,expression of the casein kinase 2β subunit gene in the kidney subjectedto diagnosis was higher than that in the non-diseased kidney used as anegative control (Table 3). Therefore, the kidney subjected to diagnosiswas diagnosed to be in the diseased condition, especially in thediseased condition involving casein kinase 2. To verify these diagnosisresults, the urinary protein excretion which is a widely used kidneyfunction marker of each rat individual diagnosed as in the diseasedcondition was measured. The urinary protein excretion of eachnon-diseased individual used as a control was also measured. As aresult, the urinary protein amounts of the individuals diagnosed as inthe kidney-diseased state were clearly larger than those of theindividuals of non-diseased state (Table 4). Thus, it was confirmed thatthe individuals diagnosed as in the kidney-diseased state were actuallyin the kidney-diseased state, and it was proved that the diagnosticmethod according to the present invention can accurately judge thediseased state of the kidney disease. TABLE 3 Results of Detection ofExpression of Casein Kinase 2 β Subunit Gene When Kidney Disease wasDiagnosed Using Expression of Casein Kinase 2 β Subunit Gene as Indexare Shown in Terms of Values. Expression Ratio Kidney (casein kinase2/G3PDH) Kidney Subjected to Diagnosis 4.5 Non-diseased Kidney 1.4

TABLE 4 Results of Measurement of Urine Protein of Individuals Diagnosedas in Kidney-diseased State Using Expression of Casein Kinase 2 βSubunit Gene as Index Individual Urine Protein (mg/day) IndividualDiagnosed as in Kidney-diseased State 146.2 Individual Not inKidney-diseased State 37.4

EXAMPLE 4

Using the expression of casein kinase 2 as an index, kidney disease wasdiagnosed. That is, using diabetic rats (Zucker strain) whichspontaneously suffered from diabetic nephropathy, expression of caseinkinase 2β subunit gene in the kidney was detected by RT-PCR method, andthe kidney disease was diagnosed using it as an index. Blood was sampledfrom the rats which spontaneously suffered from diabetic nephropathy(Zucker fa/fa strain, 6 months old, Charles River Japan, Inc.) and fromthe control rats which did not spontaneously suffered from diabeticnephropathy (Zucker Lean strain, 6 months old, Charles River Japan,Inc.), and then kidneys were isolated from the rats. From each kidney tobe subjected to diagnosis and from each control kidney which was notdiseased, RNAs were extracted by a conventional method, and cDNAs weresynthesized from 1 μg of the respective RNAs using a commerciallyavailable reverse transcription reaction kit (Invitrogen). Using 1 μleach of the synthesized cDNAs as templates, PCR was performed using theprimers for casein kinase 2α subunit and using a commercially availablePCR kit (Takara) to amplify the cDNA. Primers were prepared based on thenucleotide sequence of the full length cDNA of rat casein kinase 2βsubunit. That is, as the sense primer, 5′-ccgcggacataaagatgagt-3′ wasprepared and used, and as the antisense primer,5′-aaaccagtgccgaagtatgc-3′ was prepared and used. The PCR was carriedout by repeating 30 times the thermal cycle of 94° C. for 30 seconds,58° C. for 30 seconds and 72° C. for 40 seconds, and the reaction wasstopped finally at 4° C. Each of 10 μl aliquots of the amplified PCRproducts was electrophoresed on agarose gel, and the gel was subjectedto ethidium bromide staining, followed by measuring the expression ofthe casein kinase 2 gene. This measurement was carried out by takingphotographs of the detected casein kinase 2 so as to make image files ofthe expression of the casein kinase 2 gene, and degitalizating the imagefile using a software NIH Image on Macintosh. When the PCR was carriedout, a constantly expressing gene (a house keeping gene, G3PDH) wasdetected as an internal standard, and the results of detection of theexpression of casein kinase 2β subunit gene was compensated.

Expression of the casein kinase 2β subunit gene in the kidney subjectedto diagnosis, and the expression of the casein kinase 2β subunit gene inthe non-diseased kidney used as a control were compared. As a result,expression of the casein kinase 2 β subunit gene in the kidney subjectedto diagnosis was higher than that in the non-diseased kidney used as anegative control (Table 5). Therefore, the kidney subjected to diagnosiswas diagnosed as in the diseased condition, especially in the diseasedcondition involving casein kinase 2. To verify these diagnosis results,the blood creatinine level and the blood urea nitrogen, which are widelyused kidney function markers, of each rat individual diagnosed as in thediseased condition were measured. Those of each non-diseased individualused as a control were also measured. As a result, the blood creatininelevels of the individuals diagnosed as in the kidney-diseased state wereclearly larger than those of the individuals of non-diseased state(Table 6). Further, the blood urea nitrogen of the individuals diagnosedas in the kidney-diseased state was clearly larger than those of theindividuals of non-diseased state (Table 6). Thus, it was confirmed thatthe individuals diagnosed as in the kidney-diseased state were actuallyin the kidney-diseased state, and it was proved that the diagnosticmethod according to the present invention can accurately judge thediseased state of the kidney disease. TABLE 5 Results of Detection ofExpression of Casein Kinase 2 Gene When Kidney Disease was DiagnosedUsing Expression of Casein Kinase 2 Gene as Index are Shown in Terms ofValues. Expression Ratio Kidney (casein kinase 2/G3PDH) Kidney Subjectedto Diagnosis 3.6 Non-diseased Kidney 1.0

TABLE 6 Results of Measurement of Blood Creatinine Level and Blood UreaNitrogen of Individuals Diagnosed as in Kidney-diseased State UsingExpression of Casein Kinase 2 Gene as Index Blood Blood Urea CreatinineLevel Nitrogen Individual (mg/100 ml) (mg/100 ml) Individual Diagnosedas in Kidney- 0.89 69.0 diseased State Individual Not in Kidney-diseased0.38 18.3 State

EXAMPLE 5

Using the content of casein kinase 2 as an index, kidney disease wasdiagnosed. That is, using rats, the amounts of the casein kinase 2αsubunit protein and β subunit protein in the kidney were detected usingWestern blot method, and kidney disease was diagnosed using theseamounts.

First, to rats (Wistar-Kyoto strain, male, body weight 190 to 210 g,Charles River Japan, Inc.), rabbit anti-glomerular basement membraneantibody was intravenously administered (0.3 ml/kg) to induce nephritis.To the rats of normal group, normal rabbit serum (0.3 ml/kg) wasintravenously administered. After the induction of nephritis, all ratswere placed in metabolic cages, and urine excreted during 24 hours wascollected, and then the kidneys were isolated. From each kidney to besubjected to diagnosis and from each control kidney which was notdiseased, proteins were extracted by a conventional method, and theproteins (1 mg/ml, 20 μl) were electrophoresed (PAGERUN, ATTO, 40 mA, 84min) on 12.5% polyacrylamide gel (PAGEL, ATTO). The electrophoresedproteins were blotted (100V, 90 min) on a PVDF membrane (Millipore). Themembrane was blocked (Blocking Ace, Dainippon Pharmaceutical, 4° C., 1hour), and washed three times (10 min) with PBS containing Tween 20.Then the membrane was incubated (at room temperature for 1 hour) with ananti-casein kinase 2α subunit (originated from goat, Santa cruz,100-fold diluted) or an anti-casein kinase 2β subunit (originated fromgoat, Santa cruz, 100-fold diluted) as the primary antibody. After theincubation and after washing (10 min) the membrane 3 times with PBScontaining Tween 20, the membrane was incubated (at room temperature for1 hour) with an anti-goat IgG antibody labeled with HRP (Santa cruz,1000-fold diluted) used as the secondary antibody. After the incubation,the amounts of the casein kinase 2α subunit protein and the caseinkinase 2β subunit protein were measured, respectively, using ECL reagent(Amersham). This measurement was carried out by taking photographs ofthe detected casein kinase 2 so as to make image files of the amount ofthe casein kinase 2 protein, and degitalizating the image file using asoftware NIH Image on Macintosh.

The amount of casein kinase 2α subunit protein in the kidney subjectedto diagnosis, and the amount of casein kinase 2α subunit protein in thenon-diseased kidney used as a control were compared. As a result, theamount of casein kinase 2α subunit protein in the kidney subjected todiagnosis was higher than that in the non-diseased kidney used as anegative control (Table 7). The amount of casein kinase 2β subunitprotein in the kidney subjected to diagnosis, and the amount of caseinkinase 2β subunit protein in the non-diseased kidney used as a controlwere compared. As a result, the amount of casein kinase 2β subunitprotein in the kidney subjected to diagnosis was higher than that in thenon-diseased kidney used as a negative control (Table 8). Therefore, thekidney subjected to diagnosis was diagnosed to be in the diseasedcondition, especially in the diseased condition involving casein kinase2. To verify these diagnosis results, the urinary protein excretionwhich is a widely used kidney function marker of each rat individualdiagnosed as in the diseased condition was measured. The urinary proteinexcretion of each non-diseased individual used as a control was alsomeasured. As a result, the urinary protein amounts of the individualsdiagnosed as in the kidney-diseased state were clearly larger than thoseof the individuals of non-diseased state (Table 9). Thus, it wasconfirmed that the individuals diagnosed as in the kidney-diseased statewere actually in the kidney-diseased state, and it was proved that thediagnostic method according to the present invention can accuratelyjudge the diseased state of the kidney disease. Further, it can beeasily seen that the larger the expression amount, i.e., the content ofthe casein kinase 2 in the cells constituting the kidney, the worse thestate of the kidney disease, and the closer the expression amount, i.e.,the content, to the normal level, the closer the state of the kidneydisease to the normal state. TABLE 7 Results of Detection of Amount ofCasein Kinase 2 α Subunit When Kidney Disease was Diagnosed UsingContent of Casein Kinase α Subunit Protein as Index are Shown in Termsof Values. Protein Content Ratio of Casein Kidney Kinase 2α SubunitKidney Subjected to Diagnosis 2.2 Non-diseased Kidney 1.0

TABLE 8 Results of Detection of Amount of Casein Kinase β Subunit WhenKidney Disease was Diagnosed Using Content of Casein Kinaseβ SubunitProtein as Index are Shown in Terms of Values. Protein Content Ratio ofCasein Kidney Kinase 2β Subunit Kidney Subjected to Diagnosis 2.4Non-diseased Kidney 1.0

TABLE 9 Results of Measurement of Urine Protein of Individuals Diagnosedas in Kidney-diseased State Using Contents of Casein Kinase 2 α Subunitand β Subunit as Indices Individual Urine Protein (mg/day) IndividualDiagnosed as in Kidney-diseased State 151.7 Individual Not inKidney-diseased State 38.3

EXAMPLE 6

The relationship between casein kinase 2 protein content and enzymeactivity was analyzed. That is, each of the casein kinase 2 (Upstate)having varying protein contents (0, 10 and 20 ng protein, respectively)was incubated in 0.05 ml of a buffer (20 mM MOPS, pH7.2, 25 mMβ-glycerose phosphate, 5 mM EGTA, 1 mM sodium o-vanadate, 1 mMdithiothreitol, 15 mM MgCl₂, 0.1 mM [γ-³²P]ATP, 0.002 mCi, Amersham)containing Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu (10 nmol, 0.2 mM,Upstate) which is an enzyme substrate, at 37° C. for 10 minutes. Afterthe incubation, 0.025 ml of 40% trichloroacetic acid was added to stopthe reaction, and 0.025 ml aliquot of the reaction solution was taken onphosphocellulose paper (1 cm×1 cm). The phosphocellulose paper waswashed three times with 25 ml of 0.75% phosphoric acid for 5 minutes perwash, and then washed with 25 ml of acetone for 5 minutes, followed bymeasurement of the radioactivity of ³²P on the phosphocellulose paper byscintillation.

As a result, as shown in Table 10, the larger the casein kinase 2protein content, the higher the enzyme activity, i.e., the function ofcasein kinase 2, and it can be easily seen that the expression amount,i.e., content of casein kinase 2 protein, is closely related to theenzyme activity, i.e., function of casein kinase 2. These indicate thatthe larger the expression amount of casein kinase 2 gene of caseinkinase 2 protein, i.e., the content of the casein kinase 2 protein, theworse the state of the kidney disease, and the closer these values tothe normal levels by suppression of these, the closer the state of thekidney disease to the normal state. TABLE 10 Relationship between CaseinKinase 2 Protein Content and Enzyme Activity Amount of Protein (ng)Enzyme Activity (cpm) 0 4,511 10 46,751 20 92,203

EXAMPLE 7

This example was carried out for studying the effect of administrationof an antisense oligonucleotide against casein kinase 2α subunit, so asto confirm the utility of the present invention. To rats (Wistar-Kyotostrain, male, body weight 190 to 210 g), rabbit anti-glomerular basementmembrane antibody was intravenously administered (0.3 ml/kg) to inducenephritis. To the kidney tissues of the nephritis rats, an antisenseoligonucleotide 5′-GTAATCATCTTGATTACCCCA-3′ which selectively inhibitsexpression of casein kinase 2α subunit, or a sense oligonucleotide5′-TGGGGTATCAATCAAGATGATTAC-3′ which does not inhibit the expression ofcasein kinase 2α subunit was administered at a dose of 12 μg/day whichis said to be appropriate for the inhibition of casein kinase 2 usingAlzet pumps (0.25 μl/hour, 1002, Alzet) connected through polyethylenetubes (PE-10), continuously from one day before the induction ofnephritis to the 7th day. The nucleic acid molecules of theoligonucleotides were S-oligonucleotides containing phosphorothioatemoieties, and a cationic liquid (Polyplus transfection) was used formaking the uptake of the oligonucleotide easier. More particularly, thegroups used in the experiment were normal group (normal rats to whichthe antisense oligonucleotide against casein kinase 2α subunit and theantisense oligonucleotide were not administered, n=8), nephritis controlgroup (nephritis rats to which the antisense oligonucleotide againstcasein kinase 2α subunit and the antisense oligonucleotide were notadministered, n=8), nephritis+antisense oligonucleotide against caseinkinase 2α subunit-administered group (group of the present invention,nephritis rats to which the antisense oligonucleotide against caseinkinase 2α subunit was administered, n=4), and nephritis+senseoligonucleotide of casein kinase 2α subunit-administered group (negativecontrol group, nephritis rats to which the sense oligonucleotide ofcasein kinase 2α subunit was administered), totally four groups. On the7th day from the induction, all rats were placed in metabolic cages, andurine excreted during 24 hours was collected and the amount of the urinewas measured. The urine was then centrifuged at 3000 rpm for 15 minutes,and the urine protein in the supernatant was measured. Further, afterthe collection of urine, the kidneys were isolated, proteins wereextracted from each kidney tissue by a conventional method, and theproteins (1 mg/ml, 20 μl) were electrophoresed (PAGERUN, ATTO, 40 mA, 84min) on 12.5% polyacrylamide gel (PAGEL, ATTO). The electrophoresedproteins were blotted (100V, 90 min) on a PVDF membrane (Millipore). Themembrane was blocked (Blocking Ace, Dainippon Pharmaceutical, 4° C., 1hour), and washed three times (O min) with PBS containing Tween 20. Thenthe membrane was incubated (at room temperature for 1 hour) with ananti-casein kinase 2α subunit (originated from goat, Santa cruz,100-fold diluted) as the primary antibody. After the incubation andafter washing (10 min) the membrane 3 times with PBS containing Tween20, the membrane was incubated (at room temperature for 1 hour) with ananti-goat IgG antibody labeled with HRP (Santa cruz, 1000-fold dilutedused as the secondary antibody). After the incubation, the amounts ofthe casein kinase 2α subunit protein were measured, respectively, usingECL reagent (Amersham). This measurement was carried out by takingphotographs of the detected casein kinase 2 so as to make image files ofthe amount of the casein kinase 2 protein, and degitalizating the imagefile using a software NIH Image on Macintosh.

The results are shown in Table 11 and FIG. 2. The antisenseoligonucleotide against casein kinase 2α subunit according to thepresent invention prominently decreased the expression amount, i.e. thecontent, of the casein kinase 2α subunit protein in the kidney tissue,i.e., in the cells constituting the kidney (Table 11), and significantlydecreased the urine protein which is an important marker of the diseasedstate of kidney (FIG. 2). On the other hand, the sense oligonucleotideof casein kinase 2α subunit which was the negative control did notdecrease the expression amount, i.e. the content, of the casein kinase2α subunit protein in the kidney tissue, i.e., in the cells constitutingthe kidney (Table 11), and did not significantly decrease the urineprotein which is an important marker of the diseased state of kidney(FIG. 2). Thus, these facts give a generally recognizable theoreticalground that means and substances which decrease or inhibit theexpression of the casein kinase 2 in the kidney tissue, i.e., in thecells constituting the kidney, have actions or effects to make the stateof the kidney disease close to the normal state, so that they haveactions or effects for curing and preventing kidney diseases. Since nodifference in body weight was observed between the antisenseoligonucleotide against casein kinase 2-administered group and thenephritis control group, and between the antisense oligonucleotideagainst casein kinase 2-administered group and the negative controlgroup, and since no damage was observed in organs, it was proved thatthe antisense oligonucleotide is safely used as a therapeutic agent forkidney diseases. TABLE 11 Effects of Administration of AntisenseOligonucleotide or Sense Oligonucleotide of Casein Kinase 2 α Subunit onAmount of Casein Kinase 2 α Subunit Protein in Kidney Tissues inNephritis Rat Models Protein Amount Ratio of Casein Kidney Kinase 2 αSubunit Antisense Oligonucleotide-administered Kidney 0.45 SenseOligonucleotide-administered Kidney 1.07 No Oligonucleotide-administeredKidney 1

EXAMPLE 8

The inhibitory action of a compound, apigenin, against the enzymeactivity of casein kinase 2 was studied. Casein kinase 2 (proteinamount: 10 ng, Upstate) was incubated in 0.05 ml of a buffer (20 mMMOPS, pH7.2, 25 mM β-glycerose phosphate, 5 mM EGTA, 1 mM sodiumo-vanadate, 1 mM dithiothreitol, 15 mM MgCl₂, 0.1 mM [γ-³²P]ATP, 0.002mCi, Amersham) containing Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu (10nmol, 0.2 mM, Upstate) which is an enzyme substrate and containingapigenin at a concentration within the range of 0.1 μM to 100 μM, at 37°C. for 10 minutes. After the incubation, 0.025 ml of 40% trichloroaceticacid was added to stop the reaction, and 0.025 ml aliquot of thereaction solution was taken on phosphocellulose paper (1 cm×1 cm). Thephosphocellulose paper was washed three times with 25 ml of 0.75%phosphoric acid for 5 minutes per wash, and then washed with 25 ml ofacetone for 5 minutes, followed by measurement of the radioactivity of³²P on the phosphocellulose paper by scintillation. The ratio of theradioactivity of ³²P measured for each buffer containing the inhibitorat the respective concentration to the radioactivity of ³²P measured forthe buffer not containing apigenin was expressed in terms of % takingthe radioactivity measured for the buffer not containing apigenin as100%, and the effect against the functional activity or against theeffect of casein kinase 2 was determined.

As a result, apigenin inhibited the enzyme activity of casein kinase 2by 25% at 10 μM, and by 75% at 100 μM (FIG. 3). Thus, it is seen thatapigenin is preferred as a substance which inhibits enzyme activity ofcasein kinase 2.

EXAMPLE 9

This example was carried out to further confirm the utility of thepresent invention by studying the effect of administration of aninhibitor of casein kinase 2 to rat nephritis model using apigenin whichis preferred as a substance that inhibits enzyme activity of caseinkinase 2.

To rats (Wistar-Kyoto strain, male, body weight 190 to 210 g, CharlesRiver Japan, Inc.), rabbit anti-glomerular basement membrane antibodywas intravenously administered (0.3 ml/kg) to induce nephritis. To therats of normal group, normal rabbit serum (0.3 ml/kg) was intravenouslyadministered. Apigenin, an inhibitor of casein kinase 2, which ispreferred as a substance that inhibits the enzyme activity of caseinkinase 2, was intraperitoneally administered to each nephritis rat at adose of 20 mg/l kg body weight which is said to be appropriate for theinhibition of casein kinase, everyday from the day of induction ofnephritis to the 14th day after the induction, once a day. Theadministered apigenin solution was a 1:4 mixture of apigenin anddimethyl sulfoxide containing 0.5% (w/v) sodium carboxymethylcellulose.To the rats to which the casein kinase 2 inhibitor was not administered,the solvent alone was intraperitoneally administered. More particularly,the groups used in the experiment were normal group (normal rats towhich casein kinase 2 inhibitor was not administered, n=5), nephritiscontrol group (nephritis rats to which casein kinase 2 inhibitor was notadministered, n=5), nephritis+apigenin-administered group (group of thepresent invention, nephritis rats to which casein kinase 2 inhibitor wasadministered, n=5), totally three groups. On the 7th day from theinduction, all rats were placed in metabolic cages, and urine excretedduring 24 hours was collected and the amount of the urine was measured.The urine was then centrifuged at 3000 rpm for 15 minutes, and the urineprotein in the supernatant was measured using TP Test Wako (Wako PureChemicals).

The results are shown in FIG. 4. The casein kinase 2 inhibitor accordingto the present invention significantly decreased the urine protein whichis an important index of kidney diseases, to substantially normalizedthe state of the kidney disease. Thus, it is shown that the inhibitionof the enzyme activity of casein kinase 2 in the diseased kidney using acompoundhas an action or effort to recover the state of the kidneydisease to a state close to the normal state, so that the inhibition hasan action or effect to cure and prevent kidney diseases. In other words,it is shown that a substance having a function to inhibit casein kinase2 in the diseased kidney has an action or effect to recover the state ofthe kidney disease to a state close to the normal state, so that thesubstance has an action or effect to cure and prevent kidney diseases.That is, it is apparent that means and substance having an action toinhibit casein kinase 2 generally have an action to cure and preventkidney diseases. This indicates that kidney diseases may be cured byadministering a casein kinase 2 inhibitor. Since no difference in bodyweight was observed between the inhibitor-administered group and thenephritis control group, and since no damage was observed in organs, itwas proved that this compound is safely used as a therapeutic agent forkidney diseases.

EXAMPLE 10

This example was carried out to further confirm the utility of thepresent invention. Effect of administration of a casein kinase 2inhibitor to rat nephritis models was studied using3-methyl-1,6,8-trihydroxyanthraquinone known to have variouspharmacological actions including the action to inhibit enzyme activityof casein kinase 2 (Battistutta R et al. J Biol Chem 2000,275(38):29618-22).

To rats (Wistar-Kyoto strain, male, body weight 190 to 210 g, CharlesRiver Japan, Inc.), rabbit anti-glomerular basement membrane antibodywas intravenously administered (0.3 ml/kg) to induce nephritis. To therats of normal group, normal rabbit serum (0.3 ml/kg) was intravenouslyadministered. 3-methyl-1,6,8-trihydroxyanthraquinone (Battistutta R etal. J Biol Chem 2000, 275(38):29618-22) was intraperitoneallyadministered to each nephritis rat at a dose of 20 mg/1 kg body weightwhich was said to be appropriate for the inhibition of casein kinase,everyday from the day of induction of nephritis to the 14th day afterthe induction, once a day. The administered3-methyl-1,6,8-trihydroxyanthraquinone solution was a 1:4 mixture of3-methyl-1,6,8-trihydroxyanthraquinone and dimethyl sulfoxide containing0.5% (w/v) sodium carboxymethylcellulose. To the rats to which thecasein kinase 2 inhibitor was not administered, the solvent alone wasintraperitoneally administered. More particularly, the groups used inthe experiment were normal group (normal rats to which the casein kinase2 inhibitor was not administered, n=5), nephritis control group(nephritis rats to which the casein kinase 2 inhibitor was notadministered, n=5), nephritis+the casein kinase 2 inhibitor-administeredgroup (group of the present invention, nephritis rats to which thecasein kinase 2 inhibitor was administered, n=5), totally three groups.On the 7th day from the induction, all rats were placed in metaboliccages, and urine excreted during 24 hours was collected and the amountof the urine was measured. The urine was then centrifuged at 3000 rpmfor 15 minutes, and the urine protein in the supernatant was measuredusing TP Test Wako (Wako Pure Chemicals). On the 14th day from theinduction, the rats were placed in metabolic cages, and urine excretedduring 24 hours was collected and the amount of the urine was measured.The urine was then centrifuged at 3000 rpm for 15 minutes, and thecreatinine in the supernatant was measured using Creatinine Test Wako(Wako Pure Chemicals). After collection of the urine, blood was sampledfrom the tail vein and the sampled blood was centrifuged at 3000 rpm for15 minutes, followed by quantification of blood creatinine level usingCreatinine Test Wako (Wako Pure Chemicals). From the urinary creatininelevel and blood creatinine level, creatinine clearance was measured.Further, kidneys were isolated, fixed with 10% buffered formalin.Hematoxylin-eosin-stained specimens were prepared and microscopicallyexamined.

The results are shown in FIGS. 5, 6 and 7.3-methyl-1,6,8-trihydroxyanthraquinone known to have variouspharmacological actions including the action to inhibit enzyme activityof casein kinase 2 significantly decreased the urine protein which is animportant index of kidney diseases, and almost normalized the bloodcreatinine level and creatinine clearance. As for the pathologicalfinding of the kidney tissue, in the3-methyl-1,6,8-trihydroxyanthraquinone-administered group, improvementsin the glomeruli and in the renal tubule interstitium were observed.Thus, these show that kidney diseases may be cured by administering acasein kinase 2 inhibitor.

INDUSTRIAL AVAILABILITY

The agent for therapy and/or prevention of kidney diseases according tothe present invention may be used for the therapy and/or prevention ofkidney diseases. By the diagnostic (detection) method according to thepresent invention, kidney diseases may be diagnosed (detected)accurately and simply.

1. An agent for therapy and/or prevention of kidney diseases, comprisingas an effective ingredient a substance which inhibits casein kinase 2.2. The agent for therapy and/or prevention of kidney diseases accordingto claim 1, wherein said casein kinase 2 is originated from cellsconstituting kidney.
 3. The agent for therapy and/or prevention ofkidney diseases according to claim 1 or 2, wherein said substance is asubstance which inhibits α subunit and/or α′ subunit of casein kinase 2.4. The agent for therapy and/or prevention of kidney diseases accordingto any one of claims 1 to 3, wherein said substance is (1) a substancewhich inhibits expression of casein kinase 2, or (2) a substance whichinhibits enzyme activity of casein kinase
 2. 5. The agent for therapyand/or prevention of kidney diseases according to claim 4, wherein saidsubstance which inhibits expression of casein kinase 2 is a nucleic acidmolecule that inhibits expression of casein kinase
 2. 6. The agent fortherapy and/or prevention of kidney diseases according to claim 5,wherein said nucleic acid molecule which inhibits expression of caseinkinase 2 is an antisense oligonucleotide that targets mRNA coding forcasein kinase 2, and that can inhibit expression of casein kinase
 2. 7.The agent for therapy and/or prevention of kidney diseases according toclaim 6, wherein said antisense oligonucleotide has 12 to 50 baseshaving a sequence selected from the group consisting of coding regions,3′-untranslated regions, 5′-untranslated region, 5′ cap and intron/exonjunctions of mRNA coding for casein kinase
 2. 8. The agent for therapyand/or prevention of kidney diseases according to claim 7, wherein saidnucleic acid molecule of said antisense oligonucleotide against caseinkinase 2 is an S-oligonucleotide.
 9. The agent for therapy and/orprevention of kidney diseases according to claim 7 or 8, wherein saidnucleic acid molecule forming said antisense oligonucleotide againstcasein kinase 2 has a sequence shown in any one of SEQ ID NOs: 1-10. 10.The agent for therapy and/or prevention of kidney diseases according toclaim 9, wherein said antisense oligonucleotide against casein kinase 2has a sequence shown in SEQ ID NO:
 1. 11. The agent for therapy and/orprevention of kidney diseases according to claim 4, wherein saidsubstance which inhibits casein kinase 2 is a compound that inhibitsenzyme activity of casein kinase
 2. 12. The agent for therapy and/orprevention of kidney diseases according to claim 11, wherein saidcompound which inhibits enzyme activity of casein kinase 2 is a compoundthat inhibits enzyme activity of casein kinase 2 by not less than 30% ina range of concentration of 1 pM to 100 μM.
 13. The agent for therapyand/or prevention of kidney diseases according to claim 12, wherein saidcompound which inhibits enzyme activity of casein kinase 2 is

(wherein R¹ and R² independently represent hydrogen, hydroxy or phenylwhich may have one or more substituents selected from those representedby Formula 2, with the proviso that at least one of R¹ and R² is saidphenyl group which may have said substituent(s); R³ and R⁴ independentlyrepresent hydrogen or a substituent represented by Formula 2; and R⁵represents hydrogen, sugar residue, C₁-C₄ alkyl or acyl);—OR⁵  Formula 2 (wherein R⁵ represents the same meanings as R⁵ inFormula 2);

(wherein R and R independently represent hydrogen, halogen or C₁-C₄alkyl); TBB which is 4,5,6,7-tetrabromobenzimidazole; DRB which is5,6-dibromo-1-(β-D-ribofuranosyl)benzimidazole;

(wherein R⁸ represents phenyl which may be substituted by 1 to 3substituents selected from halogens, methyl, trifluoromethyl andhydroxy, or quinolyl, ethylmethyl sulfide, t-butylphenylthio orN-ethylphenylamino; and R⁹ represents hydroxy or amino);

(wherein n represents 1 to 3; X represents —NH— or —CONH—; and Yrepresents hydroxy, piperidine, pyrrolidine, piperazine, pyrazole ortriazole);

(wherein R¹⁰ represents 2-thienyl or phenyl which may be substituted byhalogen, methyl, ethyl or t-butyl; and R¹¹ represents —SO₂—NH₂,—SO₂—NHMe or —SO₂—NH[(CH₂)₂N(CH₃)₂]), or a pharmaceutically acceptablesalt thereof.
 14. The agent for therapy and/or prevention of kidneydiseases according to claim 13, wherein said substance which inhibitscasein kinase 2 is a compound represented by the above-described Formula1 (definitions of substituents are the same as described above) or apharmaceutically acceptable salt thereof.
 15. The agent for therapyand/or prevention of kidney diseases according to claim 14, wherein inthe Formula 1, R¹ is hydrogen or phenyl which may have one or twosubstituents selected from those shown represented by Formula 2; R² ishydrogen or hydroxy; and R⁵ is hydrogen.
 16. The agent for therapyand/or prevention of kidney diseases according to claim 15, wherein saidsubstance which inhibits casein kinase 2 is apigenin.
 17. The agent fortherapy and/or prevention of kidney diseases according to any one ofclaims 1 to 16, wherein said kidney disease is glomerular nephritis,interstitial nephritis, nephrosclerosis, diabetic nephropathy or chronicor acute renal failure.
 18. The agent for therapy and/or prevention ofkidney diseases according to any one of claims 1 to 16, wherein saidkidney disease is a nephritis other than one resulted from diabetes, orsaid kidney disease is characterized by increase in expression of caseinkinase 2 or increase in enzyme activity of casein kinase
 2. 19. Use ofsaid substance which inhibits casein kinase 2 according to any one ofclaims 1 to 18 for the production of an agent for therapy and/orprevention of kidney diseases.
 20. A method for therapy and/orprevention of kidney diseases, comprising administering an effectiveamount of said substance which inhibits casein kinase 2 according to anyone of claims 1 to 18 to a patient or an animal suffering from a kidneydisease, or to human or an animal for which prevention of a kidneydisease is desired.
 21. A method for diagnosis of kidney diseases,comprising measuring activity and/or content of casein kinase 2, and/ormeasuring expression amount of casein kinase 2 gene in a sampleseparated from body.
 22. The method according to claim 21, wherein saidsample is cells constituting kidney.
 23. The method according to claim21 or 22, wherein said casein kinase 2 is α subunit and/or α′ subunit.24. The method according to any one of claims 21 to 23, wherein saidkidney disease is glomerular nephritis, interstitial nephritis,nephrosclerosis, diabetic nephropathy or chronic or acute renal failure.25. The method according to any one of claims 21 to 23, wherein saidkidney disease is a nephritis other than one resulted from diabetes, orsaid kidney disease is characterized by increase in expression of caseinkinase 2 or increase in enzyme activity of casein kinase
 2. 26. Themethod according to any one of claims 21 to 25, comprising measuringexpression amount of casein kinase 2 gene.
 27. An oligonucleotide havinga nucleotide sequence complementary to a region in sense chain orantisense chain of casein kinase 2 gene, which is used for the methodaccording to claim
 26. 28. The oligonucleotide according to claim 27,which is a primer for gene amplification or a probe for detecting gene.